UNIVERSIDADE DE SANTIAGO DE COMPOSTELA FACULTAD DE …

UNIVERSIDADE DE SANTIAGO DE COMPOSTELA FACULTAD DE FARMACIA

Departamento de Farmacia e Tecnoloxía Farmacéutica.

NANOMEDICAMENTOS PARA EL TRATAMIENTO LOCALIZADO DE PATOLOGÍAS PULMONARES

Felipe Andrés Oyarzún Ampuero Santiago de Compostela, 2011

DOÑA DOLORES TORRES LÓPEZ Y DOÑA MARÍA JOSÉ

ALONSO FERNÁNDEZ, PROFESORA TITULAR Y

CATEDRÁTICA, RESPECTIVAMENTE, DEL DEPARTAMENTO

DE FARMACIA Y TECNOLOGÍA FARMACÉUTICA DE LA

UNIVERSIDAD DE SANTIAGO DE COMPOSTELA.

INFORMAN:

Que la presente Memoria Experimental titulada: “Nanomedicamentos

para el tratamiento localizado de patologías pulmonares”, elaborada

por el Licenciado en Farmacia Felipe Andrés Oyarzún Ampuero, ha

sido realizada bajo su dirección en el Departamento de Farmacia y

Tecnología Farmacéutica y, hallándose concluida, autorizan su

presentación a fin de que pueda ser juzgada por el tribunal

correspondiente.

Y para que conste, expiden y firman el presente certificado en Santiago

de Compostela, el 20 de Julio de 2011.

Fdo. Dolores Torres Fdo. María José Alonso

A mi familia...

"Lo escuché y lo olvidé, lo vi y lo entendí, lo hice y lo aprendí."

Confucio.

AGRADECIMENTOS

Quiero expresar mi agradecimiento a todas las personas que me han

prestado su apoyo, tanto profesional como personal, durante todo el camino

recorrido para finalizar esta tesis doctoral.

A mis directoras de tesis, las profesoras Dolores Torres y María José

Alonso, por haberme recibido y dado la gran oportunidad de realizar este

trabajo de investigación, por sus consejos, orientaciones, apoyo y gran

comprensión (incluso en importantes aspectos personales) durante todo el

tiempo que duró la parte práctica y escrita de esta tesis. Es importantísimo

hacer notar que un trabajo de similares características habría sido muy difícil

de desarrollar en mi país.

Al Ministerio de Educación de Chile y a la Xunta de Galicia, por

facilitarme el apoyo económico necesario para realizar esta tesis.

A Gustavo Rivera, mi gran amigo, por su valiosísima colaboración,

tanto teórica como práctica, desde su llegada al laboratorio.

A Begoña Seijo, por toda la ayuda prestada en su calidad de

profesora y Coordinadora del programa de doctorado. Ésta se extiende desde

antes de mi ingreso oficial al doctorado y posiblemente perdurará luego de

finalizar éste.

A todos los demás profesores, investigadores y colaboraderes del

grupo: Carmen Remuñán, Alejandro Sánchez, Marcos García, Noemí Csaba,

Francisco Goycoolea, Purificación Domínguez, Rafael Romero, etc. Todos,

absolutamente todos, tienen, además de grandes cualidades profesionales,

una inmejorable calidad humana, lo que se ha manifestado en innumerables

oportunidades y con motivo de situaciones de variada índole.

A Mabel Loza, Pepo Brea, Salvador Arines y a todos los demás

colaboradores del Departamento de Farmacología por su gran ayuda para el

desarrollo de este trabajo.

A mis compañeros y amigos del laboratorio: Giovanni, José Vicente,

Jorge, Giovanna, Patrizia, Angela, Vicky, Sonia, Celina, Pablo, Jenny, Ivana,

Yolanda, Manolo, Sascha, Ester, y todos, todos, todos, (seguro, seguro, que

se me olvida más de alguien) “le dan un espíritu y vida propia al laboratorio”,

“todos han aportado de manera tan cualitativa y cuantitativamente distinta a

los metros cuadrados que compartímos, que hacen absolutamente irrepetible

e intransferible esta etapa de mi vida”.

A todos mis amigos chilenos y gallegos con los que compartímos

tantos, tantos carretes en Santiago de Compostela (¡puchaaaaa que la

pasamos bien!), todos sabemos que hemos cultivado una amistad que

evidentemente perdurará en el tiempo y que recordaremos muy gustosamente

cuando estemos viejitos (si llegamos…).

En este punto debo agradecer mucho a los gallegos por su gran

espíritu, sencillez y capacidad para disfrutar (y los felicito por el hermoso

sitio en el que tienen la posibilidad de vivir sus vidas).

Finalmente agradezco el apoyo y comprensión de toda mi familia y

especialmente a aquellos que ya no están con nosotros, y que no podrán

disfrutar de la culminación de esta etapa de mi vida.

Gracias, muchas, muchas, muchas, gracias “sin ustedes, no lo hubiera

conseguido”.

Felipe Andrés Oyarzún Ampuero

18 de Julio, 2011.

ÍNDICE

Resumen, abstract……………………………………………….. 17

Listado de abreviaturas...………………………………………. 21

Introducción ………………………………………………………….. 25

Capitulo 1: “Nanocapsules as carriers for the transport and targeted

delivery of bioactive molecules”……………..……………………… 41

Antecedentes, Hipótesis y Objetivos ………………………………… 79

Parte I: “Desarrollo de nanosistemas híbridos de quitosano conteniendo

heparina y evaluación ex vivo de su interacción y de su actividad

antiinflamatoria sobre mastocitos"…………………………………….... 87

Capitulo 2: “Chitosan-hyaluronic acid nanoparticles loaded with

Heparin for the treatment of asthma” ………………………..……… 89

Capitulo 3: “A potential nanomedicine consisting in heparin-loaded

polysaccharide nanocarriers for the treatment of asthma”…………… 119

Discusión……….……………………………….…………………… 145

Parte II: “Desarrollo de un nuevo sistema constituído por nanocápsulas

de ácido hialurónico conteniendo docetaxel y evaluación de eficacia

antitumoral sobre cultivos celulares de cáncer de pulmón”..……..…… 165

Capitulo 4: "Hyaluronan nanocapsules: a new safe and effective

Nanocarrier for the intracellular delivery of anticancer drugs”……… 167

Discusión………..………………………………………………..... .. 199

Conclusiones…………………………………………………………… 213

Referencias…………………………………………………………..…. 217

Anexos………………………….………………………………………. 249

Anexo 1: “Chitosan-coated lipid nanocarriers for therapeutic

Applications”………………………………………………………….. 251

Anexo 2: “A new drug nanocarrier consisting of polyarginine and

hyaluronic acid”……………….………………………………………. 281

Anexo 3: Lista de Patentes....……………………………………....... 297

RESUMEN, ABSTRACT

Resumen, Abstract

17

Resumen

El objetivo de la presente memoria se ha dirigido al diseño y

evaluación de nanoestructuras para el tratamiento localizado de patologías

pulmonares. En una primera etapa, se han desarrollado nanopartículas de

quitosano, en combinación con ácido hialurónico o con carboximetil-β-

ciclodextrina, conteniendo la macromolécula hidrofílica heparina. Dichos

sistemas fueron evaluados en relación a su capacidad de mejora de eficacia

de la heparina sobre mastocitos, en el tratamiento del asma bronquial. Se

demostró por microscopía confocal de fluorescencia que los nanosistemas

eran internalizados por mastocitos de rata y, en el caso de los nanosistemas

con ciclodextrinas, se consiguió mejorar de manera significativa el efecto de

la heparina sobre la inhibición de la liberación de histamina en mastocitos.

La segunda parte del trabajo se orientó al diseño de un nuevo

nanosistema, consistente en nanocápulas de ácido hialurónico, con el fin

último de dirigirlo al tratamiento del cáncer de pulmón. Los nanosistemas

incrementaron significativamente el efecto citotóxico del antitumoral

hidrofóbico docetaxel, sobre la línea celular de cáncer de pulmón NCI-H460,

hecho que se atribuyó a la internalización de las nanocápsulas y la liberación

intracelular del docetaxel. Estos resultados resaltan el enorme interés de los

nanosistemas desarrollados para la liberación intracelular de fármacos en el

tratamiento de enfermedades pulmonares.

Resumen, Abstract

19

Abstract

The purpose of this work has been the design and evaluation of

targeted nanostructures for the treatment of lung diseases. In a first stage, we

developed nanoparticles made of chitosan combined with hyaluronic acid or

carboxymethyl-β-cyclodextrin, containing the hydrophilic macromolecule

heparin. The final aim was to explore the potential of these nanocarriers to

treat asthma. It was demonstrated that the systems were able to get inside the

mast cells and, in the case of nanosystems prepared with cyclodextrins, it was

obtained a significantly greater effect to prevent histamine release in mast

cells compared with the heparin alone.

In a second stage, we developed a new nanocarrier, named as

hyaluronic acid nanocapsules, for the intracellular delivery of hydrophobic

anticancer drugs, with potential application in lung cancer. It was shown that

these systems significantly improved the cytotoxic effect of docetaxel in the

lung cancer cell-line NCI-H460. This result was attributed to the

internalization of nanocapsules and the intracellular delivery of docetaxel. In

summary, these nanostructures hold promise as intracellular drug delivery

systems for the treatment of lung diseases.

LISTADO DE ABREVIATURAS

Listado de abreviaturas

23

Listado de abreviaturas:

AFM: Atomic force microscopy.

ANOVA: Analysis of variance.

BKC: Benzalkonium chloride,

cloruro de benzalconio.

CMβCD: Carboxymethyl-β-

cyclodextrin, carboximetil-β-

ciclodextrina.

CS: Chitosan, quitosano.

CTAB:

Hexadeciltrymethylammonium

bromide, bromuro de

hexadeciltrimetilamonio.

DCX: Docetaxel

EGF: Epidermal growth factor,

factor de crecimiento epidermal.

EPR: Enhanced permeability and

retention effect.

HA: hyaluronic acid, ácido

hialurónico.

HBSS: Hanks´ balanced salt

solution.

HPLC: High performance liquid

chromatography.

IP3: Inositoltrisphosphate,

trifosfato de inositol.

LMWH: Low molecular weight

heparin, heparina de bajo peso

molecular.

LNC: Lipid nanocapsules.

MDR: Multidrug resistance.

MTT: Tretazolium salt 3-(4,5-

dimewthylthiazol-2-yl)2,5

diphenyltetrazolium bromide.

NCs: Nanocapsules.

NMR: Nuclear magnetic

resonance.

ODN: Oligodeoxynucleotide.

PACA: Poly(alkylcyanoacrylate).

PBCA:

Poly(isobutylcyanoacrylate).

PBS: Phosphate buffered saline.

PCL: Poly-ε-caprolactone.

PEG: Polyethyleneglycol,

polietilenglicol.

PEI: Polietilenimina.

SEM: Scanning electron

microscopy.

siRNA: Small interfering RNA,

ARN pequeño de interferencia.

TEM: Transmission electron

microscopy.

TPP: Pentasodium

tripolyphosphate, tripolifosfato

pentasódico.

UFH: Unfractioned heparin,

heparina no fraccionada.

INTRODUCCIÓN

Introducción

27

INTRODUCCIÓN

1. Tratamiento localizado de patologías pulmonares

La administración pulmonar de fármacos ha experimentado un

notable impulso especialmente en la década pasada, como consecuencia de la

creciente investigación relacionada con su uso como alternativa a la

administración parenteral de macromoléculas peptídicas. Esta nueva

aplicación tiene mucho que ver con las características fisiológicas especiales

del tracto respiratorio, y la introducción de las partículas porosas, que

permitieron maximizar el acceso del fármaco a los lugares de absorción1;2.

El gran área superficial de la región alveolar, la delgada barrera

epitelial, la gran vascularización y la relativamente baja actividad proteolítica

en el espacio alveolar ofrecen grandes posibilidades a la hora de facilitar el

acceso sistémico de macromoléculas problemáticas desde el punto de vista

biofarmacéutico, como son las peptídicas3;4;5. Pero fue el descubrimiento de

que extremando la porosidad de las partículas, era posible optimizar el

acceso alveolar, lo que conllevó a la comercialización, aunque temporal, de la

insulina pulmonar, y a los diferentes estudios clínicos en marcha hoy en día

con macromoléculas inhaladas6;7;8.

Paralelamente, y como consecuencia de la nueva experimentación en

torno a la vía pulmonar, su utilización más frecuente para el tratamiento local

1 Edwards DA, Hanes J, Caponetti G, Hrkach J, Ben-Jebria A, Eskew ML, Mintzes J, Deaver D, Lotan N, Langer R. (1997). Science. 276(5320):1868-71. 2 Garcia-Contreras L, Fiegel J, Telko MJ, Elbert K, Hawi A, Thomas M, VerBerkmoes J, Germishuizen WA, Fourie PB, Hickey AJ, Edwards D. (2007). Antimicrob Agents Chemother. 51(8):2830-6. 3 Becquemin MH, Chaumuzeau JP. (2010). Rev Mal Respir. 27(8):e54-65. 4 Hohenegger M. (2010). Curr Pharm Des. 16(22):2484-92. 5 Andrade F, Videira M, Ferreira D, Sarmento B. (2011). Nanomedicine (Lond). 6(1):123-41. 6 Heinemann L. (2010). Int. J. Clin. Pract. Suppl. 166:29-40. 7 Neumiller JJ, Campbell RK. (2010). BioDrugs. 24(3):165-72. 8 Hohenegger M. (2010). Curr. Pharm. Des. 16(22):2484-92.

Introducción

28

de patologías de las vías aéreas, experimentó un considerable repunte,

abriéndose nuevas posibilidades en el tratamiento de patologías como el

asma9;10, tuberculosis11;12 o cáncer de pulmón3;13;14. A ello contribuye la

incorporación del nuevo conocimiento disponible acerca de los distintos

factores implicados, como por ejemplo, el mecanismo de acción de los

fármacos utilizados en terapias localizadas, las moléculas capaces de unirse

específicamente a receptores presentes en las células diana, o bien los nuevos

vehículos transportadores de fármacos, entre los que destacan las

nanoestructuras poliméricas, objeto de la presente Memoria.

2. Nanosistemas orientados al targeting pulmonar

El pulmón es una diana muy atractiva para la liberación de fármacos

que buscan un efecto localizado. Así, a través de una vía no invasiva, se

puede lograr una elevada concentración del principio activo, evitando el

efecto de primer paso y teniendo la posibilidad de desencadenar un rápido

inicio de acción. Este hecho, junto con el reciente desarrollo de los

nanotransportadores terapéuticos, ha generado nuevas expectativas en las

terapias localizadas (Figura 1).

9 Niven AS, Argyros G. (2003) Chest. 123(4):1254-65. 10 Barnes PJ. (2010). Trends Pharmacol. Sci. 31(7):335-43. 11 Mitchison DA, Fourie PB. (2010). Tuberculosis (Edinb). 90(3):177-81. 12 Ohashi K, Kabasawa T, Ozeki T, Okada H. (2009). J. Control Release. 135(1):19-24.. 13 Kurmi BD, Kayat J, Gajbhiye V, Tekade RK, Jain NK. (2010).Expert Opin. Drug Deliv. 7:781-794. 14 Yi D, Wiedmann T.S. (2010). J. Aerosol Med. Pulm. Drug Deliv. 23(4):181-7.

Introducción

29

Figura 1. Algunos de los sistemas nanopartículares propuestos para el targeting pulmonar (tomado de ref 13).

Los sistemas nanoparticulares ofrecen distintas ventajas potenciales

en cuanto a la optimización de:

1) adhesión a la mucosa pulmonar y modulación de la liberación del fármaco,

lo que permitiría reducir la frecuencia de dosificación, disminuir los efectos

adversos y mejorar el cumplimiento del paciente.

2) protección de fármacos macromoleculares, asegurando su liberación en

forma activa.

3) mejora de la interacción del fármaco con las células diana, favoreciendo el

acceso intracelular cuando fuese necesario, y maximizando así su eficacia15.

En la Tabla 1 se recogen distintos ejemplos de sistemas

nanoparticulares administrados por vía pulmonar para tratar diferentes

15 Manzour HM, Rhee YS, Wu X. (2009). Int. J. Nanomed. 4:299-319.

Introducción

30

afecciones localizadas en dicho órgano. Destacan en este sentido los

relacionados con el tratamiento de tuberculosis y cáncer.

En el caso de la tuberculosis, las nanoestructuras resultan

especialmente adecuadas, ya que se busca el targeting en las células diana

que son los macrófagos alveolares; por lo tanto, la captura de las

nanoestructuras por estas células, favorecería de manera rotunda el éxito del

tratamiento. Existen una serie de estudios en los que se evalúa la

administración de nanopartículas poliméricas de diversa composición,

conteniendo antibioticos (rifampicina, isoniazida y pirazinamida) a cobayas

infectadas con micobacterium tuberculosis. Dichas nanopartículas se

administraron mediante nebulización cada 10-15 días hasta un total de 3-5

dosis o bien, como control, se administraron diariamente por vía oral. En el

caso de los sistemas nanoparticulares constituídos por poli(ácido láctico-

ácido glicólico) (PLGA), se mantuvieron niveles terapéuticos plasmáticos de

los fármacos durante los 8 días postadministración, y en el pulmón estos

niveles se mantuvieron durante 11 días16. Otra interesante alternativa

consistió en modificar superficialmente las nanopartículas de PLGA con la

lectina aglutinina de germen de trigo. Dicha lectina, además de ser

mucoadhesiva, ofrece la posibilidad de que las nanopartículas interaccionen

con los receptores de esta molécula ubicados en el epitelio alveolar, lo que

permitiría prolongar aún más el control en la liberación del fármaco en el

pulmón. Los citados sistemas fueron capaces de mantener niveles

terapéuticos de los fármacos durante 14 días en el plasma y 15 días en el

pulmón17. Otra apuesta resaltable para el tratamiento de la tuberculosis

consistió en la utilización de nanopartículas de quitosano-alginato. En este

caso, se pudo apreciar igualmente que los niveles plasmáticos de los

fármacos se mantuvieron en plasma durante 14 días y en el pulmón hasta 15

16 Pandey R, Sharma A, Zahoor A, Sharma S, Khuller GK, Prasad B. (2003) J. Antimicrob. Chemother. 52(6): 981-6. 17 Sharma A, Sharma S, Khuller GK. (2004). J. Antimicrob. Chemother. 54(4): 761-6.

Introducción

31

días18. La liberación sostenida de los fármacos en el pulmón, apreciada en

todos los nanosistemas comentados, redujo significativamente la dosis total

de fármaco requerida para el tratamiento. De hecho, al final del tratamiento,

ninguno de los animales infectados presentó la enfermedad, lo que fue

comparable con lo obtenido con la dosis oral diaria.

En el caso de la administración pulmonar de nanosistemas para el

tratamiento de cáncer de pulmón, el cisplatino y sus derivados han sido

utilizados como fármacos modelo en distintas ocasiones. Estos sistemas han

sido evaluados en cuanto a sus parámetros de formulación y a su eficacia in

vitro e in vivo en diversos modelos celulares de cáncer19;20;21. Destacamos los

resultados obtenidos con nanopartículas de gelatina cargadas con cisplatino y

funcionalizadas con el factor de crecimiento epidermal (EGF). El receptor

para el EGF se sobreexpresa en distintos tipos de cáncer, especialmente en el

de células no pequeñas de pulmón22. Los autores demostraron in vitro que los

nanosistemas tenían más afinidad por células que sobrexpresan el receptor

para el EGF, y que su eficacia era mayor que la del fármaco solo o la de las

nanopartículas con cisplatino, pero sin funcionalizar. También demostraron

que la potencia de las nanopartículas funcionalizadas era superior cuando se

administraban intratumoralmente en un modelo animal de cáncer.

Finalmente, los autores administraron mediante nebulización los

nanosistemas funcionalizados y demostraron que éstos podían localizarse en

células tumorales que sobreexpresan el receptor para el EGF y alcanzar allí

altas concentraciones de fármaco23.

18 Ahmad Z, Sharma S, Khuller GK. (2005). Int .J. Antimicrob. Agents. 26(4): 298:303. 19 Cafaggi S, Russo E, Stefani R, Leardi R, Caviglioli G, Parodi B, Bignardi G, De Totero D, Aiello C, Viale M. (2007). J. Control Rel. 121(1-2):110-23. 20 Brown SD, Nativo P, Smith JA, Stirling D, Edwards PR, Venugopal B, Flint DJ, Plumb JA, Graham D, Wheate NJ. (2010). J. Am. Chem. Soc. 132(13):4678-84. 21 Paraskar AS, Soni S, Chin KT, Chaudhuri P, Muto KW, Berkowitz J, Handlogten MW, Alves NJ, Bilgicer B, Dinulescu DM, Mashelkar RA, Sengupta S. (2010). Proc. Natl. Acad. Sci. U S A. 13;107(28):12435-40. 22 Rusch V, Klimstra D, Venkatraman E, Pisters PW, Langenfeld J, Dmitrovsky E. (1997). Clin. Cancer Res. 3(4):515-22. 23 Tseng CL, Su WY, Yen KC, Yang KC, Lin FH. (2009). Biomaterials. 30(20):3476-85.

Introducción

32

Otro sistema que podemos destacar es el constituído por

nanopartículas poliméricas de poli (ácido glutámico)-dextrano recubiertas por

alcohol cetílico-tripalmitina, cargadas con 5-fluorouracilo. Dichos sistemas

se atomizaron y administraron a hámsters para evaluar su eficacia sobre

tumores de células escamosas, obteniéndose niveles efectivos de fármaco de

un modo prolongado24.

En el caso de la administración de material genético para el

tratamiento del cáncer, un trabajo interesante se refiere al tratamiento de un

modelo de metástasis pulmonar con nanosistemas constituídos por

polietilenimina y el plásmido del gen p53. Se ha encontrado que la

mutación/deleción de dicho gen está presente en la mayoría de los cánceres

de pulmón de células pequeñas y no pequeñas, por lo que transfectar dichas

células con el plásmido en cuestión resultaría favorable. Dichos sistemas

fueron nebulizados y administrados a ratas, obteniéndose reducciones muy

significativas en el tamaño y número de tumores25. También destaca el

trabajo realizado Xu y col. (2008), que diseñaron nanosistemas constituídos

por policaprolactona y polietilenimina conteniendo el RNA de interferencia

akt1 (siRNA). La proteína Akt (proteína kinasa B) es un importante regulador

de la supervivencia y proliferación celular y la amplificación de los genes que

la codifican ha sido evidenciada en varios tumores. La nebulización de estos

sistemas en ratas demostró una significativa disminución de la progresión del

tumor de pulmón, a través de la inhibición de las señales celulares

dependientes del gen Akt26.

24 Hitzman CJ, Wattenberg LW, Wiedmann TS. (2006). J. Pharm. Sci. 95(6):1196-211. 25 Densmore CL, Kleinerman ES, Gautam A, Jia SF, Xu B, Worth LL, Waldrep JC, Fung YK, T'Ang A. (2001). Cancer Gene Ther. 8(9):619-27. 26 Xu CX, Jere D, Jin H, Chang SH, Chung YS, Shin JY, Kim JE, Park SJ, Lee YH, Chae CH, Lee KH, Beck GR Jr, Cho CS, Cho MH. (2008). Am. J. Respir. Crit. Care Med. 178(1):60-73.

Introducción

33

Tabla 1: Ejemplos de sistemas nanoparticulares administrados por vía pulmonar para el tratamiento de patologías pulmonares.

Composición nanosistema Tamaño

(nm) Fármaco(s)

Especie animal

Forma de administración Respuesta biológica y

referencia PLGA, PLGA modificado con aglutinina de germen

de trigo 186-400

Rifampicina, isoniazida, pirazinamida

Cobayas Nebulización Mejora la biodisponiblidad y

permite reducir la frecuencia de dosis en tuberculosis16;17.

Quitosano-Alginato 235 Rifampicina, isoniazida,

pirazinamida Cobayas Nebulización

Mejora la biodisponiblidad y permite reducir la frecuencia de

dosis en tuberculosis18.

PLGA 213 Rifampicina Rata

Nanopartículas incluidas en microesferas de manitol

administradas en inhalador de polvo seco

Retención pulmonar prolongada del nanosistema y localización

en macrófagos alveolares27.

Quitosano 376 pDNA Ratón Instilación intratraqueal Eficacia inmunogénica contra

tuberculosis28.

Gelatina modicada con EGF

230 Cisplatino Ratón Nebulización Alta concentración del fármaco

en tumores localizados en pulmón23.

Poli(ácido glutámico)-dextrano; recubrimiento

con alcohol cetílicotripalmitina

800 5-fluorouracilo Hámsters Nebulización Se demuestra retención pulmonar

y efecto prolongado del fármaco24.

PEI No

disponible pDNA Ratón Nebulización

Reducción significativa en número y tamaño de tumores23.

27 Ohashi K, Kabasawa T, Ozeki T, Okada H. (2009). J. Control Release. 135(1):19-24. 28 Bivas-Benita M, van Meijgaarden KE, Franken KL, Junginger HE, Borchard G, Ottenhoff TH, Geluk A. (2004). Vaccine. 22(13-14):1609-15.

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34

Policaprolactona y PEI 150 siRNA Ratón Nebulización Mayor eficacia en la supresión

tumoral23.

Quitosano modificado con ácido urocánico

No disponible

Proteína de programación de muerte celular 4 (PPMC4)

Ratón Nebulización

Facilita la apoptosis, inhibe importantes vías de proliferación celular y suprime eficientemente

las vías de angiogénesis tumoral29.

PLGA 201-240 TAS-103 (fármaco

antineoplásico) Rata

Nanopartículas incluidas en microesferas de trealosa

administradas en inhalador de polvo seco

Concentración del fármaco en pulmón superior a la detectada en

plasma y superior que tras administración IV30.

PLGA-PEG 44

Oligonucleótido secuestrador del factor nuclear κB (regula la expresión de importantes

citoquinas inflamatorias)

Rata Instilación intratraqueal Atenúa el desarrollo de

hipertensión pulmonar arterial y de la remodelación arterial31.

PLGA: poli(ácido láctico-ácido glicólico); PEI:polietilenimina; PEG:polietilenglicol; EGF: factor de crecimiento epidermal; pDNA:ADN plasmídico;

siRNA:ARN pequeño de interferencia.

29 Jin H, Kim TH, Hwang SK, Chang SH, Kim HW, Anderson HK, Lee HW, Lee KH, Colburn NH, Yang HS, Cho MH, Cho CS. (2006). Mol. Cancer Ther. 5(4):1041-9. 30 Tomoda K, Ohkoshi T, Hirota K, Sonavane GS, Nakajima T, Terada H, Komuro M, Kitazato K, Makino K. (2009). Colloids Surf. B Biointerfaces. 71(2):177-82. 31 Kimura S.; Egashira K.; Chen L.; Nakano K.; Iwata E.; Miyagawa.; Tsujimoto H.; Hara K.; Morishita R.; Sueishi K.; Tominaga R.; Sunagawa K. (2009).Hypertension. 53(5):877-83.

Introducción

35

2.1. Asma bronquial y heparina

La heparina es una macromolécula que pertenece a la compleja

familia de los glucosaminoglucanos. Está compuesta por unidades

disacarídicas altamente sulfatadas (Figura 2), de tal manera que su naturaleza

polianiónica favorece la interacción con una gran variedad de proteínas que

poseen aminoácidos cargados positivamente32.

Figura 2: Estructura molecular de heparina.

De estas interacciones, la mejor caracterizada es la que lleva a la

formación del complejo heparina-antitrombina III, que posee importantes

repercusiones en el proceso de coagulación sanguínea33;34. Adicionalmente a

su actividad antiacoagulante, la heparina ha demostrado poseer interesantes

propiedades antiinflamatorias en enfermedades alérgicas. Ejemplo de ello, es

la demostrada interacción entre la heparina y el receptor intracelular de

trifosfato de inositol (IP3) en mastocitos35;36. El resultado de dicha interacción

es la inhibición de la movilización intracelular de calcio, lo que impide la

activación de mediadores intracelulares y la contracción del citoesqueleto,

previniéndose finalmente la liberación de histamina (Figura 3). Este efecto

32 Wong WS, Koh DS. (2000). Biochem. Pharmacol. 59(11):1323-35. 33 Jaques LB. (1980). Pharmacol. Rev. 31: 99-166. 34 Lindhal U, Backstrom G, Thundberg L. (1983). J. Biol. Chem. 258: 9826-9830. 35 Lucio J, D'Brot J, Guo CB, Abraham WM, Lichtenstein LM, Kagey-Sobotka A, Ahmed T. (1992). Appl. Physiol. 73(3):1093-101. 36 Ahmed T, Syriste T, Mendelssohn R, Sorace D, Mansour E, Lansing M, Abraham WM, Robinson M.J. (1994). J. Appl. Physiol. 76(2):893-901.

Introducción

36

induce a pensar el interesante rol que puede tener esta molécula en el

tratamiento del asma alérgico37;38.

Figura 3: Mecanismo de acción de heparina para prevenir la degranulación de los mastocitos.

Confirmando la información anterior, se ha demostrado en distintos

estudios que la inhalación de heparina de pesos moleculares diferentes (con y

sin actividad antiacoagulante) es efectiva para prevenir las respuestas de

broncoconstricción aguda y de hipersensibilidad bronquial, típicas de la

enfermedad asmática39;40;41;42. De hecho, se llegó a demostrar que la potencia

de las heparinas para prevenir dichas respuestas es inversamente proporcional

a su peso molecular.

Otro hallazgo interesante de la heparina, y que complementa su

utilidad para el tratamiento del asma, es su potencial para prevenir la

remodelación de las vías aéreas. Esta remodelación, mediada principalmente

37 Tyrrel DJ, Kilfeather S, Page CP. (1995). TiPS. 16:198-204. 38 Diamant Z, Page CP. (2000). Pulm. Pharmacol. Ther. 13(1):1-4. 39 Martinez-Salas J, Mendelssohn R, Abraham WM, Hsiao B, Ahmed T. (1998). J. Appl. Physiol. 84:222–228. 40 Molinari JF, Campo C, Shahida S, Ahmed T. (1998). Am. J. Respir. Crit. Care Med. 157: 887–893. 41 Campo C, Molinari JF, Ungo J, Ahmed T. (1999). J. Appl. Physiol. 86: 549–557. 42 Ahmed T, Ungo J, Zhou M, Campo C. (2000). J. Appl. Physiol. 88, 1721–1729.

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37

por la acumulación de células musculares lisas, induce un estrechamiento

excesivo de las vía aéreas y conduce a hipersensibilidad y dificultad para

respirar43. La heparina ha demostrado una alta eficacia al inhibir la

proliferación de células musculares lisas extraídas de las vías aéreas de

humanos44, bovinos45 y perros46.

2.2. Docetaxel y su vehiculización tumoral

Los taxanos (paclitaxel y docetaxel; Figura 4) son potentes agentes

quimioterápicos cuyo núcleo químico fundamental es de origen natural y se

extrae a partir de árboles del género “Taxus”. Su mecanismo de acción

consiste en promover el ensamblaje intracelular de tubulina y en inhibir la

despolimerización de los microtúbulos, impidiendo el crecimiento celular47.

Estas moléculas han contribuido de manera trascendental a la supervivencia

de pacientes con cáncer, siendo eficaces frente a un amplio rango de tumores

sólidos como el cáncer avanzado de mama, de ovarios y de células no

pequeñas de pulmón48;49;50, entre otros. Algunos estudios, como el publicado

por Jones y col. (2005), indican que el docetaxel resulta más eficaz que el

paclitaxel cuando se evalúa la sobrevida en pacientes con cáncer metastásico

de mama51.

43 Kanabar V, Hirst SJ, OʼConnor BJ, Page CP. (2005). Br. J. Pharmacol. 146, 370–377. 44 Johnson PR, Armour CL, Carey D, Black JL. (1995). Am. J. Physiol. 269, L514–L519. 45 Kilfeather SA, Tagoe S, Perez AC, Okona-Mensa K, Matin R, Page C.P. (1995). Br. J. Pharmacol. 114, 1442–1446. 46 Halayko AJ, Recto E, Sthepens NL. (1997). Can. J. Physiol. Pharmacol. 75, 917–919. 47 Abal M.; Andreu J.M.; Barasoain I. (2003). Curr Cancer Drug Targets. 3(3):193-203. 48 Rowinsky RK, Donehower RCN. (1995). Engl. J. Med. 332: 1004-1014. 49 Trudeau ME, Eisenhauer EA, Higgins BP, Letendre F, Lofters WS, Norris BD, Vandenberg TA, Delorme F, Muldal AM. (1996). J. Clin. Oncol. 14: 422-428. 50 Piccart MJ, Gore M, Huinink WTB, Vanoosterom A, Verweij J, Wanders J, Franklin H, Bayssas M, Kaye S. (1995). J. Natl. Cancer Inst. 87: 676-681. 51 Jones SE, Erban J, Overmoyer B, Budd GT, Hutchins L, Lower E, Laufman L, Sundaram S, Urba W J, Pritchard KI, Mennel R, Richards D, Olsen S, Meyers ML, Ravdin PM. (2005). J. Clin. Oncol. 2005, 23, 5542–5551.

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38

Figura 4: Estructura molecular de los taxanos (en azul y rojo se destaca la diferencia estructural entre las moléculas).

Independientemente de su potencia, ambas moléculas se caracterizan

por su elevado carácter hidrofóbico, lo que obliga a incluir agentes

solubilizantes como el Cremophor EL y Tween 80, ambos en combinación

con etanol, en las formulaciones endovenosas de paclitaxel y docetaxel,

respectivamente. Desgraciadamente, estos vehículos son responsables de

efectos secundarios severos, lo que limita la cantidad de fármaco que puede

ser administrada al paciente de modo seguro. Entre estos efectos podemos

mencionar: hipersensibilidad anafilactoide severa, neuropatía periférica,

agregación de eritrocitos y patrones anormales de lipoproteínas52;53;54.

Además de este problema de toxicidad, los taxanos son fármacos que

comparten los problemas asociados a los antitumorales, es decir, su baja

permanencia plasmática, y su biodistribución indiscriminada. Para superar

estas limitaciones, se han propuesto nuevas formulaciones que no requieren

52 Gelderblom H, Verweij J, Nooter K, Sparreboom A. (2001). Eur J Cancer. 37(13):1590-8. 53 Van Zuylen L, Verweij J, Sparreboom A. (2001).Invest. New Drugs. 19(2):125-41. 54 Engels FK, Mathot RA, Verweij J. (2007). Anticancer Drugs. 18(2):95-103.

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39

la hidrosolubilización de los fármacos, y modifican su perfil farmacocinético.

Entre ellas, destacan las basadas en nanoestructuras poliméricas, como por

ejemplo, el sistema denominado Abraxane®, constituído por nanopartículas

de albúmina conteniendo paclitaxel, la primera formulación de

nanopartículas en clínica, introducida en el año 200555. Es importante

recalcar que este hito impulsó de manera importante el desarrollo de nuevas

formulaciones que contienen taxanos, entre ellas podemos destacar:

liposomas56, lipoplejos57, nanopartículas58;59;60;61, nanocápsulas62;63 y

conjugados64;65;66. En general, todos estos nanosistemas proveen al fármaco

de una mayor citoespecificidad lo que puede verse traducido en menos

efectos no deseados y una mayor eficacia en el tratamiento.

Los sistemas nanocapsulares basados en estructuras de tipo

reservorio (núcleo de aceite recubierto por una capa polimérica), son

vehículos muy adecuados para el transporte de fármacos hidrofóbicos como

el docetaxel pues, además de obviar la necesidad de utilizar solubilizantes en

la formulación, permiten obtener una elevada eficacia de encapsulación y

55 Sparreboom A, Scripture CD, Trieu V, Williams PJ, De T, Yang A, Beals B, Figg WD, Hawkins M, Desai N. (2005). Clin. Cancer Res. 11, 4136-43. 56 Eliaz RE, Szoka FCJr. (2001). Cancer Res. 61(6):2592-601. 57 Surace C, Arpicco S, Dufaÿ-Wojcicki A, Marsaud V, Bouclier C, Clay D, Cattel L, Renoir JM, Fattal E. (2009). Mol. Pharm. 6(4):1062-73. 58 Hyung W, Ko H, Park J, Lim E, Park SB, Park YJ, Yoon HG, Suh JS, Haam S, Huh Y.M. (2008). Biotechnol. Bioeng. 99(2):442-54. 59 Pandita D, Ahuja A, Lather V, Dutta T, Velpandian T, Khar RK. (2011). Pharmazie. 66(3):171-7. 60 Hong GY, Jeong YI, Lee SJ, Lee E, Oh JS, Lee HC. (2011). Arch Pharm Res. 34(3):407-17. 61 Liu D, Wang L, Liu Z, Zhang C, Zhang N. (2010). J Biomed Nanotechnol. 6(6):675-82. 62 Lozano MV, Torrecilla D, Torres D, Vidal A, Domínguez F, Alonso MJ. (2008) Biomacromol. 9(8):2186-93. 63 Hureaux J, Lagarce F, Gagnadoux F, Rousselet MC, Moal V, Urban T, Benoit J. (2010). Pharm Res. 27(3):421-30. 64 Luo Y, Bernshaw NJ, Lu ZR, Kopecek J, Prestwich GD. (2002). Pharm Res. 19(4):396-402. 65 Rosato A, Banzato A, De Luca G, Renier D, Bettella F, Pagano C, Esposito G, Zanovello P, Bassi P. (2006). Urol. Oncol. 24(3):207-15. 66 Xin D, Wang Y, Xiang J. (2010). Pharm. Res. 27(2):380-9.

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40

modificar su perfil de distribución67;68. El Capítulo 1 de la presente tesis

doctoral es una revisión acerca de los citados sistemas, en la que se puede

obtener una visión más general y detallada de las nanocápsulas en aspectos

relacionados con su elaboración, caracterización y evaluación in vitro/in vivo.

En distintas investigaciones, se ha propuesto la utilización de

nanocápsulas para la vehiculización tumoral de docetaxel. Un ejemplo, es el

sistema propuesto por Khalid y col. (2006)69, quienes desarrollaron

nanocápsulas lipídicas recubiertas de polietilenglicol para obtener tiempos

prolongados de circulación en sangre. Este trabajo fue el primero que

demostró que la encapsulación de docetaxel en sistemas coloidales podía ser

utilizada para dirigir pasivamente el fármaco a los tejidos neoplásicos. De

hecho, las nanocápsulas demostraron una mejora en la acumulación del

fármaco en el tumor cuando se comparaban con la formulación convencional

(Taxotere®). Lozano y col. (2010)62 demostraron que la inclusión de

docetaxel en nanocápsulas recubiertas con quitosano da lugar a una rápida

captura de los sistemas en líneas celulares de cáncer de mama (MCF-7) y

pulmón (A-549) y que, tras 24 horas, el efecto sobre la viabilidad celular

obtenido con las nanocápsulas cargadas con docetaxel fue significativamente

mejor que el obtenido con el fármaco solo. Se demostró igualmente que las

nanocápsulas de quitosano mostraban, tras inyección intratumoral, un efecto

similar en la reducción del volumen tumoral al de la formulación comercial

de docetaxel, siendo un efecto más lento, pero más duradero en el período de

seguimiento del proceso70.

67 Mora-Huertas CE, Fessi H, Elaissari A. (2010). Int. J. Pharm. 385(1-2):113-42. 68 Huynh NT, Passirani C, Saulnier P, Benoit JP. (2009) Int. J. Pharm. 379(2):201-9. 69 Khalid M N, Simard P, Hoarau D, Dragomir A, Leroux J C. (2006). Pharm. Res. 23, 752–758. 70 Lozano M.V.; Torrecilla D.; Lallana E.; Vidal A.; Fernández-Megía R.; Riguera R.; Dominguez F.; Alonso M. J. and Torres D. Chitosan nanocapsules for active tumor targeting, 7th World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology, Malta, 2010.

Capitulo 1

Nanocapsules as carries for the transport and targeted delicery os

bioactive molecules

Capitulo 1

43

Introduction

Nanocapsules, first developed by Couvreur et al. 1, offer unique

opportunities with the poupose of improving the biological profile of drugs in

terms of transport across biological barriers, biodistribution and cellular

uptake. They have a vesicular organization whose internal reservoir can be

composed of aqueous or oily components, and they are surrounded by a

polymeric coating 2, 3. This reservoir system offers the possibility of great

loadings of either lipophilic or hydrophilic drugs, depending on the nature of

the liquid core (Figure ). Additionally, the core has the role of protecting the

drug from the physiological environment. Finally, the liquid nature of

nanocapsules and, thus, their elasticity, may facilitate the contact of the

nanostructures with the ephithelia and further internalization.

Polymeric Nanocapsules: Production and Characterization

Figure 1: Schematic diagram of nanocapsules containing an aqueous or oily core.

Nanocapsules as carriers for the transport and targeted delivery of bioactive molecules

44

Several methods have been developed to date for the production of

nanocapsules and the encapsulation of drugs. These methods are based on

different physicochemical principles including (i) interfacial polymerization1,

4, (ii) interfacial deposition or solvent displacement 5, (iii) phase inversion

temperature6 and (iv) polymer adsorption onto a preformed emulsion 7-9The

choice of the most appropriate materials and methods for the preparation of

the nanocapsules is critical. The specific details of the different approaches

and formulations will be described in detail in the next section.

A deep characterization of the nanocapsules is essential as there are specific

parameters, such as size, morphology and stability, which can significantly

affect their biopharmaceutical behavior 10-12.

Most of the techniques employed to characterize the morphology of

nanocapsules are based on microscopy, such as scanning electron microscopy

(SEM), atomic force microscopy (AFM) or transmission electron microscopy

(TEM). These techniques have been widely used to elucidate not only the

shape but also the size and wall thickness of nanocapsules structure. These

techniques can also be combined with other methods like freeze-fracture,

cryogenic techniques or negatively stained preparations in order to obtain

deep information on the structural organization of the different components

of the nanocapsules 3, 6, 13.

Regarding the size of the nanocapsules, several techniques can give

accurate values of diameter and wall thickness. Scattering methods are the

most recommended for obtaining accurate values of particle size

distributions. The dynamic light scattering technique, also named as photon

correlation spectroscopy, is a measurement of the dynamics of the Brownian

motion of particles, this being related to their hydrodynamic diameter. This is

a suitable method for particles with diameters between a few nanometers and

a few microns14. Recently, small angle neutron scattering has proved to be a

very powerful tool for calculating the size and the wall thickness of the

nanocapsules.15.

Capitulo 1

45

The characterization of the surface properties of the nanocapsules can

be made through the measurement of zeta potential by laser doppler

anemometry16. Nuclear Magnetic Resonance (NMR) can also be employed

for a deeper characterization of the nanocapsule surface: the hydratation and

the physical state of the shell forming polymer can be determined by cross

polarization NMR 17, while Pulsed Field Gradient NMR can be used to study

the permeability, hydratation and the mobility of the nanocapsule shell18.

A brief description of nanocapsule drug delivery systems developed to date is

presented in Table 1.

Table1: Polymers used as wall materials in nanocapsules for the delivery of different therapeutics, using varied administration routes.

Polymer Drug Drug effect Route ref

PACA NC

(water core)

Nucleic Acids Antitumor 19,28,90

Salmon calcitonin Hypocalcemic Oral 29

PACA NC Insulin Hypoglucemic Oral 52-55

Phtalocyanines Imaging i.t. 80

siRNA, ODNs Antitumor i.t. 90, 19

Cyclosporin

Pilocarpine

Immunosuppresor

Antiglaucomatous

Ocular 68,66

Eudragit Tacrolimus

Cyclosporine

Immunosuppresor

Immunosuppresor

Oral 31,56,59

PLA Indometacin,

Diclofenac

Antiinflamatory

Oral 57

PCL Spironolactone Diuretic Oral 58

Betaxolol, Carteolol,

Metipranolol

Antiglaucomatous

Ocular 63-65

Indomethacin Antiinflamatory Ocular 70,71

Cyclosporine Immunosuppresor Ocular 67,70

Chitosan Calcitonin Hypocalcemic Oral 38

Calcitonin Hypocalcemic Nasal 7

Docetaxel Antitumor 9

LNC Paclitaxel Antitumor Oral 60

Paclitaxel Antitumor i.v. 79


46

Docetaxel Antitumor i.v. 45

i.t.= intratumoral; i.v.= intravenous

Nanocapsules made of synthetic polymers

Polyacrylate nanocapsules

The first generation of nanocapsules was developed by the group of

Couvreur in the late 1970s employing poly(alkylcyanoacrylates) (PACA) as

wall material1, 4. Since then, PACA nanocapsules have been widely used in

drug delivery.

PACA nanocapsules can be prepared following two main methods:

interfacial polymerization or interfacial deposition 19, 20 (Figure 2 and 3). In

the first case, nanocapsules are formed due to the fast polymerization of the

alkylcyanoacrylate monomers at the interface of o/w or w/o emulsions

leading to the production of oil or water containing nanocapsules,

respectively. Aprotic solvents and a suitable oil/solvent ratio are necessary to

achieve an adequate yield of nanocapsules 21, 22. Oil-containing nanocapsules

prepared by this method allow the efficient encapsulation of lipophilic drugs

because of their solubility in the oily phase 23. Water soluble molecules, i.e.

insulin or calcitonin, can also be entrapped in the form of a suspension in the

oily phase 24, 25 due to the instantaneous formation of the shell around the oily

droplets. The mean diameter of the nanocapsules formed by interfacial

polymerization is normally in the range 200-350 nm. However, recently, the

possibility of reducing the size of the nanocapsules down to 100 nm was

reported, thanks to the use of the appropriate combination of surfactants 26.

Nanocapsules consisting of an aqueous core are of special interest for

the encapsulation of water-soluble molecules such as peptides 27 and nucleic

acids, including antisense oligonucleotides 28. In these cases, the

nanocapsules are formed in an external oily phase and need to be isolated and

Capitulo 1

47

resuspended in water prior to their use. Nanocapsules with aqueous core have

mean diameters ranging from 50 to 350 nm depending on the type of

surfactants used for their preparation19, 29.

Figure 2: Preparation of nanocapsules by interfacial polymerization

PACA nanocapsules can also be obtained by interfacial deposition of

a preformed polymer (Figure .3). This technique was first described by Fessi

et al.5 and is based on the spontaneous emulsification of the oil due to the

diffusion of a organic solvent, where the polymer and oil are dissolved, into

water. The nanocapsules are formed due to the precipitation of the preformed

polymer at the interface of the emulsion5, 30.The size of the nanocapsules

prepared by this method usually ranges from 150 to 300 nm.


48

Figure 3: Preparation of nanocapsules by interfacial polymer deposition following solvent displacement

Other interesting polyacrylates, also used to prepare nanocapsules,

are polymethacrylates (Eudragit®). These nanocapsules have been obtained

by interfacial deposition of the preformed Eudragit®. The interest of these

systems relies in their pH sensitive character that can be employed to

improve the stability and bioavailability of therapeutic drugs after oral

administration 31.

Polyester nanocapsules

Polyesters such as poly-ε-caprolactone (PCL), poly lactic acid (PLA)

and its copolymer poly(lactic-co-glycolic) acid (PLGA) have also been used

for the preparation of nanocapsules. To date, all polyester nanocapsules have

been prepared by the interfacial deposition of a preformed polymer following

solvent displacement 5, 13, 14. This effective and reproducible method allows

the production of polyester nanocapsules with size ranges between 100-350

nm and wall thickness of 1 to 20 nm15, 32, 33.

Capitulo 1

49

The surface properties of polyester nanocapsules can be modified in

order to reach the therapeutical purpose. For example, chitosan, a

bioadhesive polymer, can be attached to the surface of polyester

nanocapsules by incubation 16. In addition, it is possible to obtain PEG-coated

polyester nanocapsules by using the amphiphilic PEGylated copolymer, i.e.

PEG-PCL, PEG-PLA or PEG-PLGA34-37. The polymer deposition technique

leads to the orientation of the hydrophobic segment towards the oily phase

whereas the PEG portion protrudes towards the external aqueous medium.

Nanocapsules made of natural polymers

Naturally occurring polymers such as polysaccharides have also been

used for the formation of nanocapsules. Among these, chitosan has received

increasing attention for a number of years as a biomaterial for transmucosal

drug delivery. Our group described for the first time the preparation of

chitosan nanocapsules according to an interfacial deposition method slightly

modified when compared to that used for PACA or polyester nanocapsules

described above (Figure ). In this case, chitosan is incorporated into the

external aqueous phase and its deposition at the oil/water interphase occurs

because of its electrostatic interaction with the negatively charged

phosphatidylcholine, which is used as a stabilizer of the nanodroplets. 7, 8, 34

We have also proposed an alternative method which involves first, the

formation of a nanoemulsion and, the incubation of the nanoemulsion in an

aqueous solution of chitosan 7-9. This method has also been employed for the

formation of PEG-chitosan nanocapsules38. In this case, the PEG molecule

gets oriented towards the external phase due to the cationic nature of chitosan

and its natural tendency to associate to the negatively charged nanodroplets.


50

Figure 4: Preparation of nanocapsules by polymer adsorption following solvent displacement

The incubation approach has been recently proposed for the

formation of nanocapsules with a double polysaccharic wall consisting of

chitosan and lambda-carrageenan 39. In this case, the nanoemulsion was

formed by high pressure homogenization using a modified starch as

negatively charged stabilizer; then, it was incubated first in a chitosan

solution and afterwards in a lambda-carrageenan solution.

Capitulo 1

51

Lipid nanocapsules

A new generation of nanocapsules, named lipid nanocapsules, were

first prepared by the group of Benoit6, 40-42. These systems consists of an oil

core surrounded by a thick polymeric shell, made of PEG-hydroxystearate

and phosphatidylcholine. These nanocapsules can be prepared via a

novedous, solvent-free, phase inversion process (Figure ). In this process, all

the components of the system are mixed together with the aqueous phase and,

then, exposed to several cycles of heating and cooling (usually between

temperatures around 65 and 85ºC). The size and polydispersity of the

nanocapsules decrease as a function of the number and temperature cycles

and a thick interfacial layer is created with this cycling process, since the

surfactant is forced to overconcentrate at the interface of the oily droplets.43

Finally, the process is quenched at a temperature below the phase inversion

temperature (o/w emulsion), followed by addition of cold water. This fast

cooling-dilution process led to the formation of lipid nanocapsules with

particle sizes between 20 and 100 nm42. These nanocapsules showed a rigid

shell surrounding the oily core and were physically stable for at least 18

months without fusion of the dispersed oily phase41. These nanocapsules are

very versatile as they can be produced using different types of oils and lipids,

thus exhibiting high drug encapsulation efficiency values 44, 45.


52

Figure 5: Preparation of nanocapsules by phase inversion temperature.

Therapeutical applications of nanocapsules

The use of nanocapsules has been reported as a promising strategy

for improving the oral bioavailability of therapeutic molecules 46. It has been

shown that due to their colloidal size, nanocapsules are able to interact

favorably with the mucosal barrier and, simultaneously, protect the

encapsulated drug from the harsh environment of the gastrointestinal tract 46,

47. For these reasons, nanocapsules have been extensively studied as vehicles

for improving the oral bioavailability of poorly absorbed drugs such as

peptides or some lipophilic compounds, as well as for obtaining drug

controlled release48.

Capitulo 1

53

Nanocapsules for oral peptide delivery

The oral administration of peptides and proteins continue to be a

challenge because of their susceptibility to the enzymatic degradation and

their low permeability across the intestinal ephitelium. The encapsulation of

these macromolecules into polymeric nanocapsules is nowadays considered a

promising approach towards this ambitious goal49,50. An example is

represented by the nanocapsules made of mucoadhesive polymers, such as

chitosan, as described for transmucosal absorption of calcitonin 51. Chitosan

nanocapsules loaded with calcitonin were able to enhance and prolong the

systemic absorption of the drug, thus leading to an improvement of the

hypocalcemic effect (Figure ). The in vitro studies performed in the Caco-2

cells cocultured with the a model of mucus-secreting cells (HT29-M6)

suggested that chitosan nanocapsules do not cross the monolayer, but rather

they remain at the apical side of the cells 51.

Figure 6: Serum calcium levels in rats after oral administration of salmon calcitonin in an aqueous solution (sCT Sol) or encapsulated in chitosan nanocapsules (CS NC) at two different doses (250 and 500 IU/Kg), (mean ± SE; n = 6).51 Reproduced by permission of Springer.

Promising results have also been obtained with poly

(isobutylcyanoacrylate) (PBCA) nanocapsules containing calcitonin. The


54

results of the in vivo studies performed on rats indicated that these

nanocapsules allowed a great decrease of calcium levels to occur29.

On the other hand, Damgé et al. investigated the potential of PACA

nanocapsules for the oral administration of insulin52,53. Following intragastric

administration of insulin loaded nanocapsules (12.5, 25 and 50 IU of insulin

per kg) to diabetic rats (diabetes induced by the administration of 65 mg/kg

of spreptozocin), the authors observed that the nanocapsules remained intact

in simulated gastric fluid, thus ensuring a good protection of the peptide.

Moreover, the new formulation produced a significant reduction of the

glycemia (50-60%), a response that was maintained for up to 20 days. The

authors attributed this long-term effect to the adsorption of the nanocapsules

across the intestinal epithelium and the subsequent release of the

encapsulated peptide54.

More recently, other authors studied the bioavailability of orally

administered insulin loaded PBCA nanocapsules (50 IU of insulin per kg) in

diabetic rats (diabetes induced by the administration of 65 mg/kg of

spreptozocin 55. These results showed that the oral administration of

nanocapsules allows the delivery of noticeable levels of insulin into the

bloodstream in diabetic rats, however decrease in glycemia could not be

observed. The low reproducibility of the results in animal models hampered

the comprehensive analysis of the results from the different studies.

Nanocapsules were also investigated for the delivery of hydrophobic

peptides such as cyclosporine. The oral absorption of this peptide was tested

using nanocapsules made of an oily core consisting of Cremophor® or

Maisine® and surrounded by Eudragit RL® or RS®. Unfortunately, the

absolute bioavailability achieved with the nanocapsules ranged from 4 to 7.5

%, a result that is far below that observed with the marketed Neoral®

premicroemulsion (about 22%) 56. The authors related the lower cyclosporine

bioavailability to the size of the nanocapsules, more than to the constituents

of the systems, however, the low bioadhesion of the polymers used to the

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55

intestinal epithelium could also play an important role in the absorption of the

drug.

Nanocapsules for oral delivery of lipophilic low molecular weight drugs

Nanocapsules have also been used for oral delivery of low molecular

weight compounds. Anti-inflammatory agents are known to exhibit important

gastrointestinal side effects such as irritation and mucosal damage. Moreover,

they are characterized by very low water solubility, a property which makes

these good candidates for the encapsulation within oily core nanocapsules 57.

Nanocapsules made of PLA were investigated for their potential of

improving the gastrointestinal tolerance to indometacin and diclofenac57. The

encapsulation of these drugs into PLA nanocapsules led to a great reduction

of the irritation of the gastrointestinal mucosa.

The diuretic drug, spironolactone, used in premature infants to reduce

lung congestion, has also been efficiently encapsulated in PCL

nanocapsules58. Nowadays there is no commercially available oral liquid

preparation of spironolactone due to its poor water solubility and its

dissolution rate. Its incorporation into nanocapsules solved these problems,

although further pharmacokinetics studies are needed in order to fully

demonstrate their in vivo effectiveness.

The use of nanocapsules has also been proposed for the oral

administration of drugs which suffer the efflux transport, mediated by P-

glycoprotein (P-gp), across the apical membrane of the intestinal ephitelium.

This transport is known to drastically reduce the absorption of antibiotics,

antivirals, antitumorals and other drugs. Recently, it was shown that the

encapsulation of tacrolimus, an immunosuppresor agent substrate of P-gp,

into Eudragit® nanocapsules, protect the drug from the efflux transports and

increase the concentration of the drug within the cell and therefore its

bioavailability59. This evidence was observed in two animal models, rats and


56

minipigs. In addition, in these studies it was also observed that the small

lipophilic oil cores were able to enter the enterocytes and reach the lamina

propria behind the P-gp. Similar results were obtained with the encapsulation

of the antitumor drug, paclitaxel, into lipid nanocapsules. Due to the effect of

P-gp and its low water solubility, paclitaxel is currently admistered

intravenously. Following in vivo administration of lipidic nanocapsules

containing placlitaxel to rats, an increase in its absorption when compared to

that of the control was observed (Taxol®, paclitaxel dissolved in

Cremophor® and ethanol). The positive role of the nanocapsules was

attributed to two mechanisms: first, as could be expected, the presence of

lipids in the formulation increased the intestinal lymphatic transport and,

second, the entrapment of the molecule in the nanocapsules could reduce the

P-gp-mediated transport of the drug. Nevertheless, these promising results

should be taken into account cautiously due to the high interindividual

variability and need to be confirmed by further experimentation60.

Overall, nanocapsules can be considered as potential vehicles for

promoting the oral absorption of peptides and lipophilic low molecular

weight drugs. Particularly noticeable is their capacity to overcome multidrug

resistance (MDR) mechanisms, such as the P-gp efflux transport. Despite this

evidence, the validation of the efficacy of these nanosystems in large-scale

animals, in fed and fasted conditions will have to be proved in order to make

sure of their potential for clinical use.

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57

Nanocapsules as nasal drug carriers

The intranasal delivery is an attractive non-invasive route which

offers several unique advantages for peptide drugs, such as the ease of

administration, the looseness of the epithelium and the avoidance of the

hepatic first-pass metabolism. Our group has explored the potential of

chitosan nanocapsules for increasing the nasal absorption of the peptide

salmon calcitonin7. The results observed in the rat model indicated that, as

expected, the response of this peptide could be significantly enhanced and

prolonged following its association to the nanocapsules (Figure ). These

results highlight the critical role of the polymer in enhancing the transport of

the associated peptide and consequently the potential of chitosan

nanocapsules for nasal peptide delivery.

Figure 7: Serum calcium levels in rats after nasal administration of salmon calcitonin (sCT, dose: 15 IU/kg) in aqueous solution (with or without CS) or encapsulated in the control nanoemulsion (NE) or in chitosan nanocapsules (CS NC); (mean ± SE; n = 6). *Significantly different from salmon calcitonin solutions (p < 0.05). #Significantly different from nanoemulsion (p < 0.05).7 Reproduced by permission of Ed. Sante.


58

Nanocapsules as ocular drug carriers

The vast majority of intraocular diseases are treated by the instillation

of aqueous solution eye-drops in the cul-de-sac. In order to penetrate into the

eye, drugs must diffuse through different hurdles, such as the cornea, that

acts as a barrier for hydrophilic and lipophilic drugs, limiting dramatically the

intra-ocular penetration61. Another impediment is represented by the

lachrymal fluid which is continuously spread over the surface of the cornea

and is quickly drained, together with the instilled drug, into the

nasolachrymal ducts 61. In conclusion, less than 5% of the instilled drug is

able to enter into the eye 62, therefore several instillations of the drug solution

are required to obtain a sustained therapeutic effect. Importantly, drugs which

are drained into the nasolachrymal ducts can be absorbed directly into the

systemic circulation, thereby it could be possible to observe secondary effects 63-65.

The use of nanocapsules has been proposed as a strategy to increase

the penetration of lipophilic drugs into the eye by prolonging their precorneal

residence time. The strategy has been explored for a number of β-blocking

antiglaucomatous agents such as betaxolol, carteolol and metipranolol. In the

case of betaxolol and carteolol it was found that their association to PCL

nanocapsules led to a significant improvement of their pharmacological

effect (intraocular pressure) 64, 65. Additionally, in all cases, the association of

the drug to the nanocapsules resulted in a significant reduction of their side

effects 63-65. As an approach to further improving the efficacy of the

nanocapsules, Desai et al. 66 associated the antiglaucomatous drug

pilocarpine to PCL nanocapsules that were dispersed in a Pluronic® F127

gel. This formulation was more effective than the nanocapsules without the

gel or than the pilocarpine incorporated into the gel. The authors explained

the positive effect of Pluronic® F127 in terms of the ability of the gel to

increase the contact time of the nanocapsules within the ocular mucosa.

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59

Another interesting study was conducted by Calvo et al. 67 who

demonstrated an improvement in ocular absorption of cyclosporine A by

encapsulation into PCL nanocapsules. The corneal levels of this drug were 5-

fold higher than the drug formulated in oily solution, and significant

differences in the concentration of cyclosporine A were even found for up

three days. Le Bourlais et al. also studied formulations with cyclosporine A

showing that the absorption of this drug was higher when the drug was

included in PACA nanocapsules, poly(acrylic) gels, or a combination of both

compared with the drug being dispersed in oil 68. Importantly, nanocapsules

dispersed in gel did not show any toxic effect differing from the other

carriers.

In an attempt to understand the mechanism of action of nanocapsules

following topical ocular administration, our research group has conducted

several studies. In an initial study we could demonstrate by confocal

microscopy that PCL nanocapsules penetrate selectively into the corneal

epithelium by endocityc process, without cause a disruption in the cells

membrane 69. In addition, we identified that the size of the particles, but not

the inner structure or the composition, was a critical factor for their

effectiveness as drug carriers across the epithelial barrier. More specifically,

following topical instillation of different carriers containing indomethacin:

PCL nanoparticles, PCL nanocapsules, and a submicron emulsion (Figure ).

We found that all nanostructured formulations behaved significantly better

than the commercial eye drops (Indocollyre®)70 in terms of increasing the

corneal permeation of the associated drug, while PCL microparticles failed to

produce this benefit in a different study71.


60

Figure 8: Permeation of indomethacin through isolated rabbit cornea: ( ) PCL nanoparticles, ( ) PCL nanocapsules, ( ) submicron emulsion, and ( ) commercial eye drops (Indocollyre®).70 Reproduced by permission of Wiley InterScience.

In addition to the influence of the particle size, we also studied the

importance of the surface charge and composition of the nanocapsules in

their ability to work as drug carriers. More specifically, we compared the

behavior of indomethacin-loaded PCL nanocapsules with that of chitosan-

coated and poly-L-lysine-coated PCL nanocapsules following topical

instillation to rabbits. The results indicated that the chitosan-coated

nanocapsules provide better corneal drug penetration than poly-L-lysine or

uncoated nanocapsules16. Given the fact that chitosan and poly-L-lysine are

both polycationic polymer, the positive behavior of the chitosan-coated

nanocapsules could not be simply attributed to the positive surface charge but

to the specific properties of chitosan, i.e. mucoadhesive and permeability-

enhancing properties of this polymer 72.

In an attempt to explore further the effect of the surface polymer

composition in the interaction of the nanostructures with the ocular barriers,

we comparatively investigated chitosan- and PEG-coated PCL

nanocapsules34. These studies were conducted using nanocapsules loaded

with a fluorescent dye and their ocular distribution was observed by confocal

Capitulo 1

61

microscopy. Two main conclusions were extracted from this study: (i) both

formulations were internalized by the corneal epithelium; (ii) the chitosan

formulations were favourably retained in the superficial layers while the PEG

formulations were able to reach deep layers of the corneal epithelium.

All the above information indicates that nanocapsules are interesting

tools for improving the drug ocular bioavailability and reducing the systemic

side effects of drugs administered topically onto the eye. Additionally, these

results underline the importance of the particle size and surface composition

on the therapeutic efficacy of the nanocapsules.

Nanocapsules in cancer therapy

The main limitations in cancer therapies are related to their lack of

specificity and subsequent toxicity. Moreover, in many cancers, there are

specific biological barriers, such as the MDR mechanisms, which limit the

efficacy of the treatments 73, 74. Finally, from the formulation point of view,

most of the anticancer drugs suffer from poor water solubility and instability.

In this context, nanocapsule technology emerges as an important approach

for the formulation of anticancer drugs, as it offers the possibility of

incorporating hydrophobic drugs and protecting them in the biological

fluids3. The size and large surface-to-volume ratios 75 of the nanocapsules

facilitate their accumulation in the tumor by the well-known enhanced

permeability and retention effect (EPR) 76, 77 and their capacity to be

internalized by the tumor cells. Moreover, it has been shown that lipid

nanocapsules behave as a MDR-inhibiting system.78, 79.

There are a number of reports showing the advantages of

nanocapsules for specific anticancer drugs. For example, Lenaerts et al. 80

encapsulated phtalocyanines, important agents in photodynamic tumor

therapy, in poloxamer surface modified-PACA nanocapsules. They found

that the presence of some types of poloxamer significantly decreased the


62

uptake of nanocapsules by organs rich in phagocytic cells and increased the

accumulation of phtalocyanines in primary tumors. The concentration of

photosensitizers in the tumor was maximal 12 h post-administration, these

carriers allowing a 200-fold higher accumulation in the tumor.

In different reports it has been shown that lipid nanocapsules are

adequate vehicles for the delivery of taxanes. More specifically, the

encapsulation of paclitaxel into lipid nanocapsules led to a significant

concentration increase in the tumoral tissue, and significantly reduced the

tumor mass compared to the commercial product (Taxol ®) as we can see in

Figure 79. Additionally, in vivo studies in rats have shown that lipid

nanocapsules enhanced around 3-fold the oral bioavailability of the

anticancer drug, in comparison with the commercial product 60, 81. Docetaxel

is another taxane that has been encapsulated into lipid nanocapsules; these

nanocapsules showed an enhanced drug deposition in mice tumors which was

characterized by a 5-fold increase in the area under the curve of the tumor

(AUCtumor) when compared to the control formulation (Taxotere®)45.

Figure 9: In vivo effects of paclitaxel-loaded lipid nanocapsules (LNC) treatment on the growth of F98 glioma cells implanted. (C, control; Px-LNC, paclitaxel-loaded LNC; Px, Taxol only; Px + PEG-HS, Taxol with Solutol HS15 solution) *, P<0.05 (Dunnett´s test). º, P<0.05 (Fisher´s test). Statistical analysis by pairs show significant diferences on day 21 between formulations.79 Reproduced by permission of American Association for Cancer Research, Inc.

Our group has also proposed an alternative carrier for the

intracellular delivery of docetaxel consisting of oligomer chitosan

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63

nanocapsules 9. The results have shown that chitosan nanocapsules are able to

facilitate the rapid internalization of the drug into the cancer cells, leading to

a significant increase of the antiproliferative effect of the drug.

Overall, the results presented here indicate that nanocapsules

represent an alternative for the intracellular delivery of hydrophobic

anticancer drugs. This potential is related to their capacity to be internalized

by the cells and inhibit the MDR mechanisms, thus maximizing the

antitumoral drug effects.

Nanocapsules as carriers for gene therapy

The discovery of antisense oligodeoxynucleotides (ODNs) and more

recently siRNA, has opened wide perspectives in therapeutics for the

treatment of cancer, infectious and inflammatory diseases, or to block cell

proliferation and diseases caused thereby. However, the clinical use of these

molecules is limited by their poor stability in biological media and their

important hydrophilic character, which strongly limit tissular, cellular and

subcellular internalization82, 83. Besides, another disadvantage of the ODNs

and siRNA is the toxicity related with the cationic charge, and the poor

activity of these naked molecules.

A few research groups have explored the potential of nanotechnology

for the development of suitable carriers for gene delivery. Among the

different options, nanocapsule technology has been shown to offer some

specific advantages. Due to its hydrophilic character, siRNA and ODNs

molecules are usually adsorbed onto the polymeric surface of nanoparticles

or polymeric micelles84, 85, however, water containing nanocapsules can

eficiently encapsulate these molecules within its aqueous core. An interesting

method for the encapsulation of ODNs into PACA nanocapsules was that

described by Lambert et al. 28. These aqueous core-containing nanocapsules

improved the ODNs stability against enzymatic degradation and considerably


64

increased their half-life in serum in comparison with the naked molecules or

those adsorbed onto nanospheres86. Moreover, the ODNs cell uptake was

significantly improved when the molecule was included in the

nanocapsules87.

The encapsulation into the aqueous core of PBCA nanocapsules of an

antisense-siRNA (siRNA-AS) against a fusion oncogen (Fli1) overexpressed

in Ewing sarcoma, resulted in an important inhibition of tumor growth tested

in a murine model of Ewing sarcoma-related tumor (Figure )88, 89.

Figure 10: Inhibition of Erwing sarcoma fusion oncogen (EWSFli1)-expressing tumor growth in nude mice by: ○ siRNA-antisense (siRNA-AS) loaded NCs; ▲siRNA-control loaded NCs; ■, siRNA-AS naked; ♦, siRNA-control naked; ●, saline.89 Reproduced by permission of Springer.

Hillaireau et al.19 described the incorporation of ODNs to PBCA

nanocapsules. They observed that the association could be significantly

improved when ODN is associated first to a cationic polymer, such as

chitosan or poly(ethylenimine), and afterwards this complex being

encapsulated into a water containing nanocapsule. In a different work,

Bouclier et al.90 reported the encapsulation of a specific siRNA (target to

estrogen receptor alfa [ERα-siRNA]) in three different systems: PBCA

nanocapsules, PEG-PLGA nanoparticles and PEG-PCL-malic acid

nanoparticles. The in vitro studies indicated that PBCA nanocapsules showed

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65

a high efficiency in MCF-7 cancer cells, whereas the other systems showed

no antiproliferative effect in the same cancer cell lines. In a preliminary in

vivo study, these nanocapsules showed a slight decrease in tumor growth in

comparison to scramble-siRNA loaded nanocapsules or the siRNA naked90,

showing the benefits of the nanocapsules over other nanosystems for the

encapsulation of siRNA.

Conclusions

The liquid nature of nanocapsules and, thus, their fluidity and

elasticity make them ideal nanovehicles able to facilitate the contact with the

epithelia and target cells, as well as to enter intracellularly. They have unique

properties as their simplicity and their capacity of obtaining great loadings of

either lipophilic or hydrophilic drugs. Moreover, nanocapsules have shown to

be capable of inhibiting multidrug resistance cellular mechanisms, specially

important in cancer therapy. In conclusion, polymeric or lipid nanocapsules

are a promising tool for transmucosal drug delivery as well as for cancer

therapeutics, particularly for drugs which are water-insoluble and that, until

recently, have required solvents to be formulated. Concerning gene therapy,

nanocapsules emerge as an interesting approach, due to the high affinity of

nucleic acids for their water core and to the possibility of adapting these

systems to the requirements of this novel therapy.

In addition, the use of reservoir structures composed by inorganic

nanoparticles (iron, silica or gold nanoparticles, quantum dots, carbon

nanotubes, etc.) surronded by a polymer and, optionally, a targeting ligand,

represents a promising and powerful tool to enhance the biocompatibility and

the biodistribution of these nanostructures widely used in the diagnostics and

threatment of several diseases. This composite nanocapsules will be

discussed widely in following chapters.


66

Abbreviations

AFM – Atomic force microscopy

EPR – Enhanced permeability and retention effect

LNC – Lipid nanocapsules

MDR – Multidrug resistance

NCs – Nanocapsules

NMR – Nuclear magnetic resonance

ODN – Oligodeoxynucleotide

PACA – Poly(alkylcyanoacrylate)

PBCA – Poly(isobutylcyanoacrylate)

PCL – Poly-ε-caprolactone

PEG – Polyethylene glycol

PLA – Poly lactic acid

PLGA – Poly(lactic-co-glycolic) acid

SEM – Scanning electron microscopy

TEM – Transmission electron microscopy

siRNA – Small interfering RNA

siRNA – AS – siRNA antisense.

Bibliography

1. Couvreur, P., Tulkens, P., and Roland, M. (1977).Nanocapsules: a new

type of lysosomotropic carrier. FEBS Lett.84.pp 323-326.

2. Legrand, P., Barratt, G., Mosqueira, V., Fessi, H., and Devissaguet,

J.P. (1999).Polymeric nanocapsules as drug delivery systems: A

review. STP Pharm. Sci.9.pp 411-418.

3. Couvreur, P., Barratt, G., Fattal, E., Legrand, P., and Vauthier, C.

(2002).Nanocapsule technology: A review. Crit Rev Ther Drug Carrier

Syst.19.pp 99-134.

Capitulo 1

67

4. Couvreur, P., Kante, B., and Roland, M. (1979).Polycyanoacrylate

nanocapsules as potential lysosomotropic carriers: Preparation,

morphological and sorptive properties. J Pharm Pharmacol.31.pp 331-

332.

5. Fessi, H., Puisieux, F., Devissaguet, J.P., Ammoury, N., and Benita, S.

(1989).Nanocapsule formation by interfacial polymer deposition

following solvent deplacement. Int J Pharm.55.pp 25-28.

6. Heurtault, B., Saulnier, P., Pech, B., Proust, J.E., and Benoit, J.P.

(2002).A novel phase inversion-based process for the preparation of

lipid nanocarriers. Pharm Res.19.pp 875-880.

7. Prego, C., Torres, D., and Alonso, M.J. (2006).Chitosan nanocapsules:

A new carrier for nasal peptide delivery. JDDST.16.pp 331-337.

8. Prego, C., Torres, D., and Alonso, M.J. (2006).Chitosan nanocapsules

as carriers for oral peptide delivery: Effect of chitosan molecular

weight and type of salt on the in vitro behaviour and in vivo

effectiveness. J Nanosci Nanotechnol.6.pp 2921-2928.

9. Lozano, M.V., Torrecilla, D., Torres, D., Vidal, A., Dominguez, F.,

and Alonso, M.J. (2008).Highly efficient system to deliver taxanes into

tumor cells: Docetaxel-loaded chitosan oligomer colloidal carriers.

Biomacromolecules.9.pp 2186-2193.

10. Desai, M.P., Labhasetwar, V., Amidon, G.L., and Levy, R.J.

(1996).Gastrointestinal Uptake of Biodegradable Microparticles: Effect

of Particle Size. Pharm Res.13.pp 1838-1845.

11. Vila, A., Gill, H., McCallion, O., and Alonso, M.J. (2004).Transport of

PLA-PEG particles across the nasal mucosa: effect of particle size and

PEG coating density. J Control Release.98.pp 231-244.

12. Yin Win, K. and Feng, S. (2005).Effects of particle size and surface

coating on cellular uptake of polymeric nanoparticles for oral delivery

of anticancer drugs. Biomaterials.26.pp 2713-2722.


68

13. Quintanar-Guerrero, D., Allemann, E., Doelker, E., and Fessi, H.

(1998).Preparation and characterization of nanocapsnles from

preformed polymers by a new process based on emulsification-

diffusion technique. Pharm Res.15.pp 1056-1062.

14. Moinard-Checot, D., Chevalier, Y., Briancon, S., Beney, L., and Fessi,

H. (2008).Mechanism of nanocapsules formation by the emulsion-

diffusion process. J Colloid Interface Sci.317.pp 458-468.

15. Rube, A., Hause, G., Mader, K., and Kohlbrecher, J. (2005).Core-shell

structure of Miglyol/poly(D,L-lactide)/Poloxamer nanocapsules

studied by small-angle neutron scattering. J Control Release.107.pp

244-252.

16. Calvo, P., Vila-Jato, J.L., and Alonso, M.J. (1997).Evaluation of

cationic polymer-coated nanocapsules as ocular drug carriers. Int J

Pharm.153.pp 41-50.

17. Guinebretiere, S., Briancon, S., Lieto, J., Mayer, C., and Fessi, H.

(2002).Study of the emulsion-diffusion of solvent: Preparation and

characterization of nanocapsules. Drug Dev Res.57.pp 18-33.

18. Wohlgemuth, M. and Mayer, C. (2003).Pulsed field gradient NMR on

polybutylcyanoacrylate nanocapsules. J Colloid Interface Sci.260.pp

324-331.

19. Hillaireau, H., Le Doan, T., Chacun, H., Janin, J., and Couvreur, P.

(2007).Encapsulation of mono- and oligo-nucleotides into aqueous-

core nanocapsules in presence of various water-soluble polymers. Int J

Pharm.331.pp 148-152.

20. Vauthier, C. and Bouchemal, K. (2008).Methods for the Preparation

and Manufacture of Polymeric Nanoparticles. Pharm Res.1-34.

21. Gallardo, M., Couarraze, G., Denizot, B., Treupel, L., Couvreur, P.,

and Puisieux, F. (1993).Study of the mechanisms of formation of

nanoparticles and nanocapsules of polyisobutyl-2-cyanoacrylate. Int J

Pharm.100.pp 55-64.

Capitulo 1

69

22. Puglisi, G., Fresta, H.T.M., Giammona, G., and Ventura, C.A.

(1995).Influence of the preparation conditions on

poly(ethylcyanoacrylate) nanocapsule formation. Int J Pharm.125.pp

283-287.

23. Fresta, M., Cavallaro, G., Giammona, G., Wehrli, E., and Puglisi, G.

(1996).Preparation and characterization of polyethyl-2-cyanoacrylate

nanocapsules containing antiepileptic drugs. Biomaterials.17.pp 751-

758.

24. Aboubakar, M., Puisieux, F., Couvreur, P., Deyme, M., and Vauthier,

C. (1999).Study of the mechanism of insulin encapsulation in

poly(isobutylcyanoacrylate) nanocapsules obtained by interfacial

polymerization. J Biomed Mater Res.47.pp 568-576.

25. Lowe, P.J. and Temple, C.S. (1994).Calcitonin and insulin in

isobutylcyanoacrylate nanocapsules: Protection against proteases and

effect on intestinal absorption in rats. J Pharm Pharmacol.46.pp 547-

552.

26. Vauthier, C., Labarre, D., and Ponchel, G. (2007).Design aspects of

poly(alkylcyanoacrylate) nanoparticles for drug delivery. J Drug

Target.15.pp 641-663.

27. Watnasirichaikul, S., Davies, N.M., Rades, T., and Tucker, I.G.

(2000).Preparation of biodegradable insulin nanocapsules from

biocompatible microemulsions. Pharm Res.17.pp 684-689.

28. Lambert, G., Fattal, E., Pinto-Alphandary, H., Gulik, A., and Couvreur,

P. (2000).Polyisobutylcyanoacrylate nanocapsules containing an

aqueous core as a novel colloidal carrier for the delivery of

oligonucleotides. Pharm Res.17.pp 707-714.

29. Vranckx, H., Demoustier, M., and Deleers, M. (1996).A new

nanocapsule formulation with hydrophilic core: Application to the oral

administration of salmon calcitonin in rats. Eur J Pharm

Biopharm.42.pp 345-347.


70

30. Al Khouri Fallouh, N., Roblot-Treupel, L., and Fessi, H.

(1986).Development of a new process for the manufacture of

polyisobutylcyanoacrylate nanocapsules. Int J Pharm.28.pp 125-132.

31. Nassar, T., Rom, A., Nyska, A., and Benita, S. (2008).A novel

nanocapsule delivery system to overcome intestinal degradation and

drug transport limited absorption of P-glycoprotein substrate drugs.

Pharm Res.25.pp 2019-2029.

32. Guinebretiere, S., Briançon, S., Fessi, H., Teodorescu, V.S., and

Blanchin, M.G. (2002).Nanocapsules of biodegradable polymers:

Preparation and characterization by direct high resolution electron

microscopy. Mater Sci Eng C.21.pp 137-142.

33. Cauchetier, E., Deniau, M., Fessi, H., Astier, A., and Paul, M.

(2003).Atovaquone-loaded nanocapsules: Influence of the nature of the

polymer on their in vitro characteristics. Int J Pharm.250.pp 273-281.

34. De Campos, A.M., Sanchez, A., Gref, R., Calvo, P., and Alonso, M.J.

(2003).The effect of a PEG versus a chitosan coating on the interaction

of drug colloidal carriers with the ocular mucosa. Eur J Pharm

Sci.20.pp 73-81.

35. Mosqueira, V.C.F., Legrand, P., Gulik, A., Bourdon, O., Gref, R.,

Labarre, D., and Barratt, G. (2001).Relationship between complement

activation, cellular uptake and surface physicochemical aspects of

novel PEG-modified nanocapsules. Biomaterials.22.pp 2967-2979.

36. Mosqueira, V.C.F., Legrand, P., Morgat, J.L., Vert, M., Mysiakine, E.,

Gref, R., Devissaguet, J.P., and Barratt, G. (2001).Biodistribution of

long-circulating PEG-grafted nanocapsules in mice: Effects of PEG

chain length and density. Pharm Res.18.pp 1411-1419.

37. Bouclier, C.l., Moine, L., Hillaireau, H., Marsaud, V.r., Connault, E.,

Opolon, P., Couvreur, P., Fattal, E., and Renoir, J.-M.

(2008).Physicochemical Characteristics and Preliminary in Vivo

Capitulo 1

71

Biological Evaluation of Nanocapsules Loaded with siRNA Targeting

Estrogen Receptor Alpha. Biomacromolecules.9.pp 2881-2890.

38. Prego, C., Torres, D., Fernandez-Megia, E., Novoa-Carballal, R.,

Quinoa, E., and Alonso, M.J. (2006).Chitosan-PEG nanocapsules as

new carriers for oral peptide delivery: Effect of chitosan pegylation

degree. J Control Release.111.pp 299-308.

39. Preetz, C., Rube, A., Reiche, I., Hause, G., and Mader, K.

(2008).Preparation and characterization of biocompatible oil-loaded

polyelectrolyte nanocapsules. Nanomedicine.4.pp 106-114.

40. Heurtault, B., Saulnier, P., Pech, B., Benoit‚t, J.P., and Proust, J.E.

(2003).Interfacial stability of lipid nanocapsules. Colloids Surf B

Biointerfaces.30.pp 225-235.

41. Heurtault, B., Saulnier, P., Pech, B., Proust, J.E., and Benoit, J.P.

(2003).Physico-chemical stability of colloidal lipid particles.

Biomaterials.24.pp 4283-4300.

42. Heurtault, B., Saulnier, P., Pech, B., Venier-Julienne, M.C., Proust,

J.E., Phan-Tan-Luu, R., and Benoit, J.P. (2003).The influence of lipid

nanocapsule composition on their size distribution. Eur J Pharm

Sci.18.pp 55-61.

43. Anton, N., Gayet, P., Benoit, J.P., and Saulnier, P. (2007).Nano-

emulsions and nanocapsules by the PIT method: An investigation on

the role of the temperature cycling on the emulsion phase inversion. Int

J Pharm.344.pp 44-52.

44. Babu Dhanikula, A., Mohamed Khalid, N., Lee, S.D., Yeung, R.,

Risovic, V., Wasan, K.M., and Leroux, J.C. (2007).Long circulating

lipid nanocapsules for drug detoxification. Biomaterials.28.pp 1248-

1257.

45. Khalid, M.N., Simard, P., Hoarau, D., Dragomir, A., and Leroux, J.C.

(2006).Long circulating poly(ethylene glycol)-decorated lipid


72

nanocapsules deliver docetaxel to solid tumors. Pharm Res.23.pp 752-

758.

46. Prego, C., Garcia, M., Torres, D., and Alonso, M.J.

(2005).Transmucosal macromolecular drug delivery. J Control

Release.101.pp 151-162.

47. Csaba, N., Garcia-Fuentes, M., and Alonso, M.J. (2006).The

performance of nanocarriers for transmucosal drug delivery. Expert

Opin Drug Deliv.3.pp 463-478.

48. Pinto Reis, C., Neufeld, R.J., Ribeiro, A.J., and Veiga, F.

(2006).Nanoencapsulation II. Biomedical applications and current

status of peptide and protein nanoparticulate delivery systems.

Nanomedicine.2.pp 53-65.

49. Alonso, M.J. (2004).Nanomedicines for overcoming biological

barriers. Biomed Pharmacother.58.pp 168-172.

50. des Rieux, A., Fievez, V., Garinot, M., Schneider, Y.J., and Preat, V.

(2006).Nanoparticles as potential oral delivery systems of proteins and

vaccines: A mechanistic approach. J Control Release.116.pp 1-27.

51. Prego, C., Fabre, M., Torres, D., and Alonso, M.J. (2006).Efficacy and

mechanism of action of chitosan nanocapsules for oral peptide

delivery. Pharm Res.23.pp 549-556.

52. Damge, C., Michel, C., Aprahamian, M., and Couvreur, P. (1988).New

approach for oral administration of insulin with polyalkylcyanoacrylate

nanocapsules as drug carrier. Diabetes.37.pp 246-251.

53. Damge, C., Vranckx, H., Balschmidt, P., and Couvreur, P.

(1997).Poly(alkyl cyanoacrylate) nanospheres for oral administration

of insulin. J Pharm Sci.86.pp 1403-1409.

54. Pinto-Alphandary, H., Aboubakar, M., Jaillard, D., Couvreur, P., and

Vauthier, C. (2003).Visualization of insulin-loaded nanocapsules: In

vitro and in vivo studies after oral administration to rats. Pharm

Res.20.pp 1071-1084.

Capitulo 1

73

55. Cournarie, F., Auchere, D., Chevenne, D., Lacour, B., Seiller, M., and

Vauthier, C. (2002).Absorption and efficiency of insulin after oral

administration of insulin-loaded nanocapsules in diabetic rats. Int J

Pharm.242.pp 325-328.

56. Ubrich, N., Schmidt, C., Bodmeier, R., Hoffman, M., and Maincent, P.

(2005).Oral evaluation in rabbits of cyclosporin-loaded Eudragit RS or

RL nanoparticles. Int J Pharm.288.pp 169-175.

57. Staniscuaski Guterres, S., Fessi, H., Barratt, G., Puisieux, F., and

Devissaguet, J.P. (1995).Poly(D,L-Lactide) nanocapsules containing

non-steroidal anti-inflammatory drugs: Gastrointestinal tolerance

following intravenous and oral administration. Pharm Res.12.pp 1545-

1547.

58. Limayem Blouza, I., Charcosset, C., Sfar, S., and Fessi, H.

(2006).Preparation and characterization of spironolactone-loaded

nanocapsules for paediatric use. Int J Pharm.325.pp 124-131.

59. Nassar, T., Rom, A., Nyska, A., and Benita, S. (2009).Novel double

coated nanocapsules for intestinal delivery and enhanced oral

bioavailability of tacrolimus, a P-gp substrate drug. J Control

Release.133.pp 77-84.

60. Peltier, S., Oger, J.M., Lagarce, F., Couet, W., and Benoit, J.P.

(2006).Enhanced oral paclitaxel bioavailability after administration of

paclitaxel-loaded lipid nanocapsules. Pharm Res.23.pp 1243-1250.

61. Barar, J., Javadzadeh, A.R., and Omidi, Y. (2008).Ocular novel drug

delivery: Impacts of membranes and barriers. Expert Opin Drug

Deliv.5.pp 567-581.

62. Lang, J.C. (1995).Ocular drug delivery conventional ocular

formulations. Adv Drug Deliv Rev.16.pp 39-43.

63. Losa, C., Marchal-Heussler, L., Orallo, F., Vila-Jato, J.L., and Alonso,

M.J. (1993).Design of new formulations for topical ocular


74

administration: Polymeric nanocapsules containing metipranolol.

Pharm Res.10.pp 80-87.

64. Marchal-Heussler, L., Fessi, H., Devissaguet, J.P., Hoffman, M., and

Maincent, P. (1992).Colloidal drug delivery systems for the eye. A

comparison of the efficacy of three different polymers:

Polyisobutylcyanoacrylate, polylactic-co-glycolic acid, poly-epsilon-

caprolactone. STP Pharma Sciences.2.pp 98-104.

65. Marchal-Heussler, L., Sirbat, D., Hoffman, M., and Maincent, P.

(1993).Poly(ε-caprolactone) nanocapsules in carteolol ophthalmic

delivery. Pharm Res.10.pp 386-390.

66. Desai, S.D. and Blanchard, J. (2000).Pluronic® F127-based ocular

delivery system containing biodegradable polyisobutylcyanoacrylate

nanocapsules of pilocarpine. Drug Delivery: J Delivery and Targeting

Therapeutic Agents.7.pp 201-207.

67. Calvo, P., Sanchez, A., Martinez, J., Lopez, M.I., Calonge, M., Pastor,

J.C., and Alonso, M.J. (1996).Polyester nanocapsules as new topical

ocular delivery systems for cyclosporin A. Pharm Res.13.pp 311-315.

68. Le Bourlais, C.A., Chevanne, F., Turlin, B., Acar, L., Zia, H., Sado,

P.A., Needham, T.E., and Leverge, R. (1997).Effect of cyclosporine A

formulations on bovine corneal absorption: Ex-vivo study. J

Microencapsulation.14.pp 457-467.

69. Calvo, P., Thomas, C., Alonso, M.J., Vila-Jato, J.L., and Robinson,

J.R. (1994).Study of the mechanism of interaction of poly(E-

caprolactone) nanocapsules with the cornea by confocal laser scanning

microscopy. Int J Pharm.103.pp 283-291.

70. Calvo, P., Vila-Jato, J.L., and Alonso, M.J. (1996).Comparative in

vitro evaluation of several colloidal systems, nanoparticles,

nanocapsules, and nanoemulsions, as ocular drug carriers. J Pharm

Sci.85.pp 530-536.

Capitulo 1

75

71. Calvo, P., Alonso, M.J., Vila-Jato, J.L., and Robinson, J.R.

(1996).Improved Ocular Bioavailability of Indomethacin by Novel

Ocular Drug Carriers. J Pharm Pharmacol.48.pp 1147-1152.

72. Alonso, M.J. and Sanchez, A. (2003).The potential of chitosan in

ocular drug delivery. J Pharm Pharmacol.55.pp 1451-1463.

73. Gottesman, M.M., Fojo, T., and Bates, S.E. (2002).Multidrug

resistance in cancer: Role of ATP-dependent transporters. Nat Rev

Cancer.2.pp 48-58.

74. Ehdaie, B. (2008).Application of Nanotechnology in Cancer Research:

Review of Progress in the National Cancer Institute's Alliance for

nanotechnology.

75. McNeil, S.E. (2005).Nanotechnology for the biologist. J Leukoc

Biol.78.pp 585-594.

76. Iyer, A.K., Khaled, G., Fang, J., and Maeda, H. (2006).Exploiting the

enhanced permeability and retention effect for tumor targeting. Drug

Discov Today.11.pp 812-818.

77. Greish, K. (2007).Enhanced permeability and retention of

macromolecular drugs in solid tumors: A royal gate for targeted

anticancer nanomedicines. J Drug Target.15.pp 457-464.

78. Brigger, I., Dubernet, C., and Couvreur, P. (2002).Nanoparticles in

cancer therapy and diagnosis. Adv Drug Deliv Rev.54.pp 631-651.

79. Garcion, E., Lamprecht, A., Heurtault, B., Paillard, A., Aubert-

Pouessel, A., Denizot, B., Menei, P., and Benoit, J.P. (2006).A new

generation of anticancer, drug-loaded, colloidal vectors reverses

multidrug resistance in glioma and reduces tumor progression in rats.

Mol Cancer Ther.5.pp 1710-1722.

80. Lenaerts, V., Labib, A., Chouinard, F., Rousseau, J., Ali, H., and Van

Lier, J. (1995).Nanocapsules with a reduced liver uptake: Targeting of

phthalocyanines to EMT-6 mouse mammary tumor in vivo. Eur J

Pharm Biopharm.41.pp 38-43.


76

81. Lacoeuille, F., Hindre, F., Moal, F., Roux, J., Passirani, C., Couturier,

O., Cales, P., Le Jeune, J.J., Lamprecht, A., and Benoit, J.P. (2007).In

vivo evaluation of lipid nanocapsules as a promising colloidal carrier

for paclitaxel. Int J Pharm.344.pp 143-149.

82. Fattal, E. and Bochot, A. (2008).State of the art and perspectives for

the delivery of antisense oligonucleotides and siRNA by polymeric

nanocarriers. Int J Pharm.364.pp 237-248.

83. Li, X. and Chan, W.K. (1999).Transport, metabolism and elimination

mechanisms of anti-HIV agents. Adv Drug Deliv Rev.39.pp 81-103.

84. Nakada, Y., Fattal, E., Foulquier, M., and Couvreur, P.

(1996).Pharmacokinetics and biodistribution of oligonucleotide

adsorbed onto poly(isobutylcyanoacrylate) nanoparticles after

intravenous administration in mice. Pharm Res.13.pp 38-43.

85. Schwab, G., Chavany, C., Duroux, I., Goubin, G., Lebeau, J., Helene,

C., and Saison-Behmoaras, T. (1994).Antisense oligonucleotides

adsorbed to polyalkylcyanoacrylate nanoparticles specifically inhibit

mutated Ha-ras-mediated cell proliferation and tumorigenicity in nude

mice. Proc Natl Acad Sci U S A.91.pp 10460-10464.

86. Lambert, G., Fattal, E., Pinto-Alphandary, H., Gulik, A., and Couvreur,

P. (2001).Polyisobutylcyanoacrylate nanocapsules containing an

aqueous core for the delivery of oligonucleotides. Int J Pharm.214.pp

13-16.

87. Lambert, G., Bertrand, J.R., Fattal, E., Subra, F., Pinto-Alphandary, H.,

Malvy, C., Auclair, C., and Couvreur, P. (2000).EWS Fli-1 antisense

nanocapsules inhibits Ewing sarcoma-related tumor in mice. Biochem

Biophys Res Commun.279.pp 401-406.

88. Toub, N., Bertrand, J.R., Malvy, C., Fattal, E., and Couvreur, P.

(2006).Antisense oligonucleotide nanocapsules efficiently inhibit

EWS-Fli1 expression in a Ewing's sarcoma model.

Oligonucleotides.16.pp 158-168.

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77

89. Toub, N., Bertrand, J.R., Tamaddon, A., Elhamess, H., Hillaireau, H.,

Maksimenko, A., Maccario, J., Malvy, C., Fattal, E., and Couvreur, P.

(2006).Efficacy of siRNA nanocapsules targeted against the EWS-Fli1

oncogene in Ewing sarcoma. Pharm Res.23.pp 892-900.

90. Bouclier, C., Moine, L., Hillaireau, H., Marsaud, V., Connault, E.,

Opolon, P., Couvreur, P., Fattal, E., and Renoir, J.M.

(2008).Physicochemical characteristics and preliminary in vivo

biological evaluation of nanocapsules loaded with siRNA targeting

estrogen receptor alpha. Biomacromolecules.9.pp 2881-2890.

ANTECEDENTES, HIPÓTESIS Y OBJETIVOS

Antecedentes, hipótesis y objetivos

81

ANTECEDENTES

1. Las nanoestructuras poliméricas administradas por inhalación, han

demostrado ser capaces de proporcionar niveles terapéuticos de fármacos

durante períodos prolongados, al potenciar el acceso de éstos hacia las dianas

localizadas a nivel pulmonar71;72;73;74;75.

2. Algunos polisacáridos mucoadhesivos, como el quitosano y el ácido

hialurónico, ofrecen interesantes posibilidades como constituyentes de

nanoestructuras capaces de interaccionar con el epitelio pulmonar y

maximizar su período de contacto76;77. Ambos polímeros pueden, además,

formar nanoestructuras híbridas gracias a la interación iónica de sus cargas

opuestas78;79, lo que aporta ventajas en cuanto al perfil de seguridad ofrecido

por el quitosano79.

3. La combinación del quitosano con ciclodextrinas en nanopartículas, aporta

mejoras a las propiedades de los nanosistemas constituídos únicamente por el

polisacárido, al disminuir posibles alteraciones de la función barrera del

epitelio y ofrecer mayor protección a las macromoléculas encapsuladas80;81.

71 Pandey R, Sharma A, Zahoor A, Sharma S, Khuller GK, Prasad B. (2003). J. Antimicrob. Chemother. 52(6): 981-6. 72 Sharma A, Sharma S, Khuller GK. (2004). J. Antimicrob. Chemother. 54(4): 761-6. 73 Ahmad Z, Sharma S, Khuller GK. (2005). Int .J. Antimicrob. Agents. 26(4): 298:303. 74 Hitzman CJ, Wattenberg LW, Wiedmann TS. (2006). J Pharm Sci. 95(6):1196-211. 75 Ohashi K, Kabasawa T, Ozeki T, Okada H. (2009). J. Control Release. 135(1):19-24. 76 Yamamoto H, Kuno Y, Sugimoto S, Takeuchi H, Kawashima Y. (2005). J Control Release. 102(2):373-81. 77 Liu XB, Ye JX, Quan LH, Liu CY, Deng XL, Yang M, Liao YH. (2008). Eur J Pharm Biopharm. 78 de la Fuente M, Seijo B, Alonso MJ. (2008). Macromol Biosci. 8(5):441-50. 79 de la Fuente M, Seijo B, Alonso MJ. (2008). Invest. Ophthalmol. Vis. Sci. 49(5):2016-24. 80 Teijeiro-Osorio D, Remuñán-López C, Alonso MJ. (2009). Biomacromol. 10(2):243-9. 81 Chen Y, Siddalingappa B, Chan PH, Benson HA. (2008). Biopolymers. 90(5):663-70.


82

4. La heparina es una macromolécula que ofrece un potencial terapéutico

para el tratamiento del asma bronquial, al prevenir la desgranulación de los

mastocitos82;83. Este potencial se ve limitado por su acceso restringido al

interior celular84, donde se encuentran localizados los receptores.

5. El docetaxel constituye un tratamiento antitumoral de elección, sin

embargo su extremada hidrofobicidad obliga a la inclusión de disolventes en

su formulación endovenosa, que dan lugar a importantes reacciones de

sensibilización que se suman a los efectos secundarios provocados por el

propio citostático85;86.

6. Nuestro grupo de investigación ha demostrado el potencial de

nanocápsulas de polisacáridos y poliaminoácidos catiónicos como

transportadores intracelulares de antitumorales hidrofóbicos, como el

docetaxel, consiguiendo vehículos eficaces sin necesidad de la inclusión de

solventes tóxicos87;88.

7. Se ha demostrado que en distintos tumores sólidos, entre ellos varios de

pulmón, hay sobreexpresión del receptor CD-44, que es la diana endógena

del ácido hialurónico89;90. La incorporación de ácido hialurónico en diferentes

82 Tyrrel DJ, Kilfeather S, Page CP. (1995). TiPS. 16:198-204. 83 Diamant Z, Page CP. (2000). Pulm. Pharmacol. Ther. 13(1):1-4. 84 Motlekar NA, Youan BB. (2006). J. Control Release. 113(2):91-101. 85 Engels FK, Mathot RA, Verweij J. (2007). Anticancer Drugs. 18(2):95-103. 86 Ten Tije AJ, Verweij J, Loos WJ, Sparreboom A. (2003). Clin. Pharmacokinet. 42(7), 665-85. 87 Lozano MV, Torecilla D, Torres D, Vidal A, Dominguez F, Alonso MJ. (2008). Biomacromol. 9, 2186-2193. 88 Lozano MV, Lollo G, Brea J, Torres D, Loza MI, Alonso MJ. Polyarginine nanocapsules: a new platform for intracellular drug delivery (submitted). 89 Penno MB, August JT, Baylin SB, Mabry M, Linnoila RI, Lee VS, Croteau D, Yang XL, Rosada C. (1994). Cancer Res. 54(5):1381-7. 90 Tran TA, Kallakury BV, Sheehan CE, Ross JS. (1997). Hum Pathol. 28(7):809-14.


83

tipos de transportadores ha confirmado la especificidad del targeting hacia el

receptor CD-4491;92;93;94.

HIPÓTESIS

1.- El desarrollo de nanoestructuras constituídas por polisacáridos

mucoadhesivos como el quitosano y/o el ácido hialurónico, puede constituir

una estrategia adecuada para tratar localmente patologías que cursan a nivel

pulmonar, como el asma bronquial o el cáncer de pulmón. El éxito de esta

estrategia residirá fundamentalmente en favorecer la accesibilidad de los

fármacos a las células diana, a la vez que prolongar su contacto con las

mismas y potenciar su captura intracelular.

2.- La combinación del quitosano con ciclodextrinas en sistemas

nanoparticulares es una alternativa que puede aportar ventajas al sistema puro

en su administración pulmonar, en lo que se refiere a mejora de su perfil de

seguridad, promoción de su captura intracelular, y protección de la

macromolécula encapsulada.

3.- El diseño de un nuevo sistema constituído por nanocápsulas de ácido

hialurónico puede resultar de interés para la vehiculización pulmonar del

antitumorales hidrofóbicos. El nuevo nanosistema contendrá en su núcleo

oleoso la molécula activa, mientras que el ácido hialurónico potenciará la

interacción con células tumorales que sobreexpresan receptores CD-44 en su

superficie.

91Akima K, Ito H, Iwata Y, Matsuo K, Watari N, Yanagi M, Hagi H, Oshima K, Yagita A, Atomi Y, Tatekawa I. (1996). J Drug Target. 1996;4(1):1-8. 92 Auzenne E, Ghosh SC, Khodadadian M, Rivera B, Farquhar D, Price RE, Ravoori M, Kundra V, Freedman RS, Klostergaard J. (2007). Neoplasia. 9(6):479-86. 93 Eliaz RE, Szoka FCJr. (2001). 61(6), 2592-601. 94 Peer D, Margalit R. (2004). Neoplasia 6, 343-353.


84

OBJETIVOS

El objetivo de esta Tesis se ha dirigido a evaluar el potencial que

presentan distintos nanotransportadores para vehiculizar y promover el efecto

intracelular de fármacos tan distintos, como la macromolécula hidrofílica

heparina o el antitumoral hidrofóbico docetaxel, en células diana pulmonares.

Este objetivo se cubrirá a través de las siguientes etapas:

1.- Desarrollo de nanosistemas híbridos de quitosano conteniendo

heparina y evaluación ex vivo de su interacción y de su actividad

antiinflamatoria sobre mastocitos.

Esta parte de la memoria se ha dirigido en primer lugar a optimizar

los nanosistemas de quitosano-ácido hialurónico y quitosano-ciclodextrinas

en cuanto a su contenido en heparina, para asegurar la producción del efecto

antiasmático. En segundo lugar, se estudió la capacidad de los nanosistemas

para interaccionar y ser internalizados en mastocitos extraídos de rata.

Finalmente, se ha evaluado el potencial de estos vehículos para inhibir la

liberación de histamina por parte de los mastocitos.

Los resultados de este apartado se recogen en los capítulos experimentales 2

y 3.

2.- Desarrollo de un nuevo sistema constituído por nanocápsulas de ácido

hialurónico conteniendo docetaxel y evaluación de su eficacia

antitumoral sobre cultivos celulares de cáncer de pulmón.


85

Para poner a punto el nuevo nanosistema, se llevó a cabo la

optimización del proceso de recubrimiento con el polímero aniónico,

evaluando sus características tras la incorporación de distintos tensoactivos

catiónicos en el núcleo oleoso. Se determinó finalmente la eficacia

antitumoral de las nanocápsulas conteniendo docetaxel sobre el modelo

celular de cáncer de pulmón NCI-H460.

Los resultados de este apartado se recogen en el capítulo experimental 4.

PARTE I: DESARROLLO DE NANOSISTEMAS HÍBRIDOS DE

QUITOSANO CONTENIENDO HEPARINA Y EVALUACIÓN EX VIVO

DE SU INTERACCIÓN Y DE SU ACTIVIDAD ANTIINFLAMATORIA

SOBRE MASTOCITOS.

Capitulo 2

Chitosan-hyaluronic acid nanoparticles loaded with heparin for the

treatment of asthma

Capitulo 2

91

Abstract

The purpose of this study was to produce mucoadhesive nanocarriers

made from chitosan (CS) and hyaluronic acid (HA), and containing the

macromolecular drug heparin, suitable for pulmonary delivery. For the first

time, this drug was tested in ex-vivo experiments performed in mast cells, in

order to investigate the potential of the heparin-loaded nanocarriers in

antiasthmatic therapy. CS and mixtures of HA with unfractionated or low-

molecular-weight heparin (UFH and LMWH, respectively) were combined to

form nanoparticles by the ionotropic gelation technique. The resulting

nanoparticles loaded with UFH were between 162 and 217 nm in size, and

those prepared with LMWH were 152 nm. The zeta potential of the

nanoparticle formulations ranged from +28.1 to +34.6 mV, and in selected

nanosystems both types of heparin were associated with a high degree of

efficiency, which was approximately 70%. The nanosystems were stable in

phosphate buffered saline (PBS), pH 7.4, for at least 24 h, and released

10.8% of UFH and 79.7% of LMWH within 12 h of incubation. Confocal

microscopy experiments showed that fluorescent heparin-loaded CS-HA

nanoparticles were effectively internalized by rat mast cells. Ex-vivo

experiments aimed at evaluating the capacity of heparin to prevent histamine

release in rat mast cells indicated that the free or encapsulated drug exhibited

the same dose-response behaviour.

Keywords: Nanoparticles; Chitosan; Hyaluronic Acid; Heparin; Asthma;

Mast Cells; Histamine.

Capitulo 2

93

Introduction

Although mast cells produce a variety of lipid mediators,

chemokines, cytokines and enzymes that can interact with airway smooth

muscle cells to cause hyperresponsiveness (Page et al., 2001; Robinson,

2004), they are the only endogenous source of heparin in mammals, which

plays a protective role by limiting inflammation and airway remodelling

(Page, 1991). Heparin is released on degranulation of mast cells (Green et al.,

1993) and inhibits the proliferation of smooth muscle cells isolated from the

airways of several species including humans (Johnson et al., 1995), bovines

(Kilfeather et al., 1995) and dogs (Halayko et al., 1997).

Furthermore, several studies have demonstrated that the inhalation of

high, medium and low-molecular-weight heparin (with or without

anticoagulant activity) is effective in preventing acute bronchoconstrictor

responses and airway hyperresponsiveness, with the potency of these types of

heparin being inversely proportional to their molecular weight (Martinez-

Salas et al., 1998; Molinari et al., 1998; Campo et al., 1999; Ahmed et al.,

2000). This effect was attributed to the capacity of heparin to prevent mast

cell degranulation. Interestingly, ultra-low-molecular-weight heparin was

also effective in the treatment of late airway responses (pre or post antigen

challenge); the effect was independent of the anticoagulant activity of the

heparin and was mediated by an unknown biological action (Molinari et al.,

1998; Ahmed et al., 2000). This is convincing evidence of the potential role

of heparin in asthma therapy.

With the aim of enhancing the potential role of heparin in the

treatment of asthma, we propose encapsulating this macromolecule in

selected nanocarriers capable of positively interacting with mast cells, be

internalized by these cells and released the encapsulated heparin in a

controlled manner, thereby also preventing the possible degradation of the

drug by enzymatic attack in the airways.

Chitosan-hyaluronic acid nanoparticles loaded with heparin for the treatment of asthma

94

CS is a natural, non-toxic, biodegradable polycationic

polysaccharide. We, and other groups, have previously used the polymer to

elaborate different nanocarriers (Garcia-Fuentes et al., 2005; Köping-

Höggård et al., 2005; Prego et al., 2005; De la Fuente et al; 2008a); these

nanosystems have been shown, among other advantages, to prolong its

residence time at the target site of absorption. These results were mainly

attributed to the capacity of the polymer to interact with the negatively

charged cell surfaces.

HA is a natural, non-toxic, biodegradable polysaccharide that is

distributed widely throughout the human body, mainly in the connective

tissue, eyes, intestine and lungs. Several ex-vivo studies have demonstrated

that particulate HA systems have beneficial effects on the mucociliary

transport rate in airways, due to the mucoadhesivity of the polymer (Prichtard

et al., 1996; Lim et al., 2000). It has also been found that HA has a discreet

hypoproliferative effect on proliferating airway smooth muscle cells

(Kanabar et al., 2005). This may also indicate that HA alone, or in synergy

with heparin (Johnson et al., 1995), may be useful in preventing narrowing of

the airway in asthmatic patients.

Taking into account this information and the previous experience of

our group on the development of CS-HA nanoparticles loaded with

hydrophilic and hydrophobic macromolecules (De la Fuente et al., 2008b),

the present study aimed to combine the virtues of CS and HA in the

development of heparin-loaded nanoparticles, intended for pulmonary

administration. Finally, the interaction between these nanosystems and mast

cells will be investigated, and their potential for preventing rat mast cell

degranulation evaluated, to our knowledge, for the first time.

Capitulo 2

95

Materials and methods

Materials

Ultrapure chitosan hydrochloride salt (CS; UP CL 113, molecular

weight ~125 KDa and degree of acetylation 14%) was purchased from

Pronova Biopolymer AS (Oslo, Norway). Sodium hyaluronate ophthalmic

grade (HA, molecular weight ~165 KDa) was a gift from Bioiberica

(Barcelona, Spain). Unfractionated heparin sodium salt (UFH, molecular

weight ~18 KDa, 202 USP units/mg), low-molecular-weight heparin sodium

salt (LMWH, molecular weight ~4 KDa, 53 USP units/mg) and pentasodium

tripolyphosphate (TPP) were purchased from Sigma Aldrich (Madrid, Spain).

All other solvents and chemicals were of the highest commercially available

grade.

Preparation of heparin-loaded CS-HA nanoparticles

CS-HA nanoparticles loaded with heparin were prepared according to

the procedure previously developed by our group (De la Fuente et al., 2008a).

Nanoparticles were spontaneously obtained by ionotropic gelation between

the positively charged amino groups of CS and the negatively charged HA,

TPP and heparin. Briefly, 3.5 mL of a mixture of an aqueous solution of HA

(0.17-0.34 mg/mL), TPP (0.06 mg/mL) and UFH (0.29-0.46 mg/mL) or

LMWH (0.4 mg/mL) were added to 3.5 mL of a solution of CS (0.11% w/v,

pH 4.9) under magnetic stirring at room temperature. Magnetic stirring was

maintained for 10 min to enable complete stabilization of the system. The

nanoparticles were transferred to Eppendorf tubes and isolated by

centrifugation in 20 μL of a glycerol bed (16000×g, 30 min, 25º C).

Supernatants were collected and the nanoparticles were then resuspended in

ultrapure water by shaking on a vortex mixer.


96

The production yield of the systems was obtained by centrifugation

of fixed volumes of the nanoparticle suspensions (16000×g, 30 min, 25° C),

without a glycerol bed. The supernatants were discarded and the sediments

were freeze-dried. The yield was calculated as follows:

100components theofamount Total

lesnanopartic ofWeight Yield ×=

Physicochemical characterization of heparin-loaded CS-HA

nanoparticles

The size and zeta potential of the colloidal systems were determined

by photon correlation spectroscopy and laser Doppler anemometry, with a

Zetasizer Nano-ZS (Malvern Instruments, United Kingdom). Each batch was

analyzed in triplicate.

Morphological examination of the nanoparticles was performed by

transmission electron microscopy (TEM) (CM12 Philips, Netherlands). The

samples were stained with 1% w/v phosphotungstic acid for 10 sec.,

immobilized on copper grids with Formvar® and dried overnight for viewing

by TEM.

Association efficiency and drug loading of heparin-loaded CS-HA

nanoparticles

The association efficiencies of the selected formulations were

determined after isolation of nanoparticles by centrifugation, as described in

Section 2.2. The amount of unbound heparin in the supernatant was

determined by a colorimetric method (Stachrom® Heparin, Diagnostica

Stago, France).

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97

The association efficiency of heparin and the drug loading were

calculated as follows:

100drug ofamount Total

drug unbound ofAmount - drug ofamount Total efficiencyn Associatio ×=

100 lesnanopartic ofWeight

drug unbound ofAmount - drug ofamount TotalLoading Drug ×=

Stability study of heparin-loaded CS-HA nanoparticles in different

media

Selected nanoparticle formulations were prepared and centrifuged in

the presence of glycerol. Nanoparticles were tested for their stability taking

into account the change in size of nanoparticles and possible precipitations in

different media at 37º C, including: Hanks´ balanced salt solution (HBSS) at

pH 6.4 and 7.4, and phosphate buffered saline (PBS), at pH 7.4 (for

composition of these solutions, see below). Nanoparticles were incubated in

these media and samples were collected at several time intervals (0, 1, 3, 5,

10 and 24 h), and the size distribution of the nanoparticles was measured by

photon correlation spectroscopy.

The composition of HBSS was: 137 mM NaCl, 5.4 mM KCl, 0.25 mM

Na2HPO4, 0.44 mM KH2PO4 and 4.2 mM NaHCO3.

The composition of PBS was: 137 mM NaCl, 2.7 mM KCl, 1.4 mM

NaH2PO4 and 1.3 mM Na2HPO4.

In vitro heparin release studies from CS-HA nanoparticles

Heparin release studies were performed by incubating 0.1 mg of the

selected nanoparticles in 1 mL of PBS (pH 7.4) at 37º C. The samples were


98

centrifuged at appropriate time intervals (1, 5 and 12 h), and the amount of

heparin released was evaluated with the heparin kit described above. The

concentration of heparin was quantified and calculated by interpolation from

the corresponding standard curve.

Study of interaction of fluorescent heparin-loaded CS-HA nanoparticles

with rat mast cells by confocal microscopy

Fluorescein labelling of CS

Chitosan was labelled with fluorescein following a slight

modification of the method described by De Campos et al. (2004). The

covalent attachment of fluorescein to CS was by the formation of amide

bonds between primary amino groups of the polymer and the carboxylic acid

groups of fluorescein. Briefly, 250 mg of CS was dissolved in 25 mL of

water, and 10 mg of fluorescein (Sigma Aldrich, Spain) was dissolved in 1

mL of ethanol. These solutions were then mixed, and EDAC (1-ethyl-3-

(dimethylaminopropyl) carbodiimide hydrochloride) (Sigma Aldrich, Spain)

was added to a final concentration of 0.05 M, to catalyze the formation of

amide bonds. The reactive mixture was incubated under permanent magnetic

stirring for 12 h in the dark, at room temperature. The resulting conjugate

was finally isolated by dialysis for 72 h (cellulose dialysis tubing, pore size

12400 Da; Sigma Aldrich, Spain) against demineralised water, and freeze-

dried. The pH of fluorescent CS was adjusted to the same value as the raw

CS solution (pH 4.9) with HCl, for the preparation of fluorescent

nanoparticles.

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99

Preparation of fluorescent heparin-loaded CS-HA nanoparticles

Fluorescent nanoparticles were prepared according to the same

procedure described in 2.2. The selected mass distribution for the preparation

of fluorescent nanoparticles was: 4 mg of fluorescent CS, 0.6 mg of HA, 0.21

mg of TPP and 1.4 mg of UFH.

Confocal laser scanning microscopy study

An aqueous solution (50 μL) containing 0.3 mg of isolated

fluorescent UFH-loaded CS-HA nanoparticles was incubated with 450 μL of

a suspension of mast cells (10x103 cells/100 μL) in Umbreit (for

composition, see below) containing 0.05% w/v of BSA. The mixture was

incubated for 2 h at 37 °C, and the cells were then separated by centrifugation

(10 min, 200xg) and discarding the supernatants. Two hundred μL of

Umbreit+BSA solution (at 4 °C) were then added to the cell pellet. The pellet

was resuspended and centrifuged again to extract the non-internalized

nanoparticles. This procedure was repeated once more. Mast cells were fixed

for 5 minutes in paraformaldehyde (2% w/v, 100 μL) and washed 3 times

with the Umbreit+BSA solution, by centrifugation. Two hundred μL of a

Bodipi® phalloidin solution (Invitrogen, USA) were added to the cell pellet

and the cells were incubated for 30 min at room temperature. The cells were

washed 3 times (Umbreit+BSA) by centrifugation, the supernatant was

discarded, and the pellet was resuspended in 20 μL of the Umbreit+BSA

solution. The resuspended sample was placed on the surface of a positively

charged microscope slide (Superfrost Ultra Plus, Menzel-Glaser, Irland) and

dried at room temperature overnight. The sample was prepared in

Vectashield medium (Vector, USA) for visualization by confocal microscopy

(CLSM, Zeiss 501, Germany) (all of the described procedures were carried


100

out in darkness to prevent the loss of the fluorescent signal from the

nanoparticles and mast cells).

The composition of Umbreit saline solution was: 1.2 mM MgSO4, 1.2 mM

NaPO4H2, 22.85 mM NaHCO3, 5.94 mM KCl, 1 mM CaCl2, 119 mM NaCl

and 0.1% glucose.

Ex vivo studies with rat mast cells: Inhibition of histamine release by

heparin-loaded CS-HA nanoparticles

Rat mast cell purification and viability

Mast cells were obtained by lavage of pleural and peritoneal cavities

of female Sprague–Dawley rats (400–800 g) with Umbreit saline solution,

following procedures similar to those described in other studies (Lago et al.,

2001; Buceta et al., 2008). The suspension obtained from each rat was

centrifuged at 100xg for 5 min (4 ºC) and suspended in a final volume of 1

mL of Umbreit containing 0.05% w/v of BSA. Purification was carried by

centrifugation on 4 mL of an isotonic Percoll gradient at 600 g for 10 min (4

ºC). The mast cells were washed twice with the Umbreit+BSA solution and

maintained at 4 °C in this solution until use. Mast cells were quantified by

toluidin blue staining (95% purity) and the viability assessed by trypan blue

staining (the procedure is described below).

Trypan blue staining procedure: Mast cell viability studies were

carried out by trypan blue staining in an inverted microscope, as described by

Lago et al. (2001). This involved visual counting of the stained cells in the

five fields of a counting chamber. The percentage of viability was calculated

with the following formula:

100 cellsmast ofnumber Totalfields five thefrommean Arithmetic cells stained blueTrypan ×=

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101

In order to test the mast cell viability after contact with heparin-

loaded nanoparticles, the same procedure was used, and the UFH or LMWH-

loaded CS-HA nanoparticles added to the rat mast cell suspension (1x105

cells per test tube). The tested dose of nanoparticles was equivalent to 200

μg/mL of UFH or LMWH.

Measurement of histamine release in rat mast cells

Rat mast cells (1x105 cells per test tube) were pre-warmed at 37 ºC

(10 min) in BSA-free Umbreit saline solution containing the UFH or LMWH

solutions or the nanoparticles loaded with UFH or LMWH. Histamine release

from mast cells was then initiated by incubating the cells with 100 µM of

compound 48/80 (Sigma Aldrich, Spain) for 20 min at 37 ºC. The cells were

then centrifuged at 1100xg for 3 min at 4 ºC, and two aliquots (100 µl) of the

supernatants were collected in a 96-well microplate. The rest of the

supernatants were discarded and the pellets were resuspended in 500 μl of 0.1

mM HCl, sonicated for 1 min and centrifuged at 1100 x g for 6 min. Two

aliquots of 100 μL of the supernatants were collected for residual histamine

determination. Histamine was assayed fluorometrically, as described by Lago

et al. (2001); briefly, 80 µL NaOH 1 M were added to 100 µL of the sample,

then 50 µl phthaldialdehyde 0.04% w/v were added to each well and plate

was incubated for 4 min at 25° C. After this time, 50 µl of 3 M HCl were

added and fluorescence was measured within 20 min, at excitation and

emission wavelengths of 360 nm and 465 nm respectively, in a Tecan Ultra

Evolution reader (Tecan, Switzerland).

Data analysis for measurement of histamine release in rat mast cells


102

Results were expressed as percentage of the total histamine released

after stimulation with compound 48/80. The results were corrected for

spontaneous histamine release in the absence of any chemical and under the

same conditions. The equation used for the calculation was

HR=[(S−ER)/(S+P−ER)]×100, where HR is the percentage histamine

release; S, supernatant fluorescence; ER, fluorescence of spontaneous release

supernatants and P, pellet fluorescence.

IC50 values were obtained by fitting the data with non-linear regression, with

Prism 2.1 software (GraphPad, San Diego, CA).

Statistical analysis

The statistical significance of the differences between formulations

was determined by application of two-way analysis of variance (ANOVA)

followed by a two-tailed paired Student’s test. Differences were considered

significant at p<0.05.

Results and discussion

Preparation and characterization of heparin-loaded CS-HA

nanoparticles

Nanoparticles loaded with heparin were prepared by the ionotropic

gelation technique. The ability of CS to form a gel after contact with

polyanions by promoting inter and intramolecular linkages (Calvo et al.,

1997) enables the formation of the nanoparticles. In this case, an ionic

interaction occurs between the positively charged CS and the negatively

charged HA, heparin and the polyanion TPP. The ionic gelation process is

extremely simple and involves mixing two aqueous phases at room

temperature.

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103

When the nanoparticles loaded with UFH were prepared, it was

necessary to establish the best ratio between components that enabled

formation and also adequate isolation of the nanosystems. The size,

polydispersity index, zeta potential and appearance of the tested formulations

are shown in Table 1. In general, it is possible to argue that when the amount

of polyanions was too low (relative to CS), nanoparticles could not be

formed, or that the quantity of the formed nanoparticles was too low.

Nanoparticles with different characteristics were obtained when greater

amounts of polyanions were used. However, if the amount of polyanions was

too high, it was impossible to isolate the particles, because the nanosystems

were not resuspendable or, in extreme cases, precipitation occurred. In

addition, when the amount of polyanions was higher, slight decreases in the

positive zeta potential values were observed. This may be caused by

increased shielding of free positively charged groups of CS.

All the resulting nanosystems ranged in size from 162 to 217 nm;

polydispersity values were between 0.11-0.45 and the positive zeta potential

ranged from +28.1 to +34.6 (Table 1). This positive zeta potential indicates

that the surface of the nanosystems is preferably composed by CS. Among

the tested formulations, we selected those formed by the following mass

distribution for subsequent studies: 4 mg of CS, 0.6 mg of HA, 0.21 mg of

TPP and 1.4 mg of UFH. This formulation was able to encapsulate the

highest amount of UFH tested, showed reasonable polydispersity, and high

turbidity (related to a higher production yield).


104

Table 1: Physicochemical properties of the nanoparticles prepared with different ratios of CS- HA-TPP-heparin (mean ± S.D., n=3).

Amount (mg) CS-HA-TPP-

heparin

Size (nm)

Polydispersity Index

Zeta potential

(mV) Appearance

4-1.2-0.21-1.0a 201 ± 24 0.22 – 0.36 +32.1 ± 1.6 Medium turbidity

4-1.2-0.21-1.2a 217 ± 30 0.23 – 0.35 +28.1 ± 0.9 High turbidity 4-1.2-0.21-1.4a Not resuspendable --- --- --- 4-1.2-0.21-1.6a Precipitation --- --- ---

4-0.6-0.21-1.2a 162 ± 17 0.11 – 0.30 +34.6 ± 0.6 Medium turbidity

4-0.6-0.21-1.4a 193 ± 32 0.24 – 0.45 +32.5 ± 1.7 High turbidity 4-0.6-0.21-1.5a Not resuspendable --- --- --- 4-0.6-0.21-1.4b 152 ± 10 0.17 – 0.27 +33.0 ±1.3 Low turbidity

a= UFH; b= LMWH

The loading capacity, association efficiency and yield of the selected

formulation are shown in Table 2. The association efficiency was 72.3% and

therefore 1.01 mg of UFH (of the initial 1.4 mg) formed nanoparticles. The

drug loading was about 34%, with the remaining mass corresponding to CS,

HA and TPP.

The same formulation prepared with LMWH was similar in size, zeta

potential and association efficiency, but showed lower values of

polydispersity and yield and higher drug loading (see Tables 1 and 2).

Interestingly, the drug loading of the formulation with LMWH is

approximately twice that of the formulation with UFH, and it is possible that

LMWH may induce greater displacement of the anionic molecules (HA and

TPP) from the nanoparticles and, consequently, lead to a lower yield.

Table 2: Loading characteristics and yield of selected CS-HA nanoparticles containing UFH or LMWH (mean ± S.D., n=3).

Amount (mg) CS-HA-TPP-heparin

Loading capacity (%)

Association Efficiency (%)

Yield (%)

4-0.6-0.21-1.4a 33.6 ±1.2 72.3 ± 2.7 49.0 ± 1.2 4-0.6-0.21-1.4b 60.6 ± 0.3 69.7 ± 7.6 24.9 ± 4.3

a = UFH; b = LMWH

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105

Considering that the only difference between the prepared

formulations was the type of heparin, these changes should be attributed, on

one hand, to the different molecular weight (~18 KDa for UFH and ~4 KDa

for LMWH) and, on the other hand, to possible chemical differences between

UFH and LMWH, associated with very different values of anticoagulant

activities (202 and 53 USP units/mg, respectively).

The TEM micrographs shown in Figures 1a and 1b indicate that the

selected nanosystems loaded with UFH or LMWH were spherical.

Interestingly, the systems with UFH appeared denser in the center than at the

surface, and differed from those containing LMWH, which appeared more

homogeneous.

Figure 1: Electron transmission micrographs of selected CS-HA nanoparticles containing UFH (a) or LMWH (b).

Stability studies of heparin-loaded CS-HA nanoparticles

Determination of nanoparticle colloidal stability under conditions

similar to those used for cell culture is crucial for future studies. Therefore,

the stability of the selected systems was investigated in media usually used

for cell culture studies. These media included: HBSS (pH 7.4), HBSS (pH

6.4) and PBS (pH 7.4). The stability of the selected nanoparticles was better

in media of pH 7.4, and was maximal for PBS, where the size was

maintained for up to 24 h (Fig. 2) (the stability in HBSS (pH 6.4), is not

shown because the nanosystems aggregated immediately). The explanation


106

for this difference between PBS (pH 7.4) and HBSS (pH 7.4), may be related

to the different composition of these media (HBSS contains CO3-2

ions and a

concentration of PO4-2 ions eight times lower than in PBS). This may directly

affect the hydration shell of the counterions located at the surface of the

nanoparticles, as well as the structure of water surrounding the systems. In

PBS these effects appeared to result in an increase in repulsive hydration

forces, and hence, greater stability. Interestingly, the zeta values of UFH and

LMWH-loaded nanoparticles was slightly negative (-10.5±2 mV) throughout

all the stability studies in which HBSS (pH 7.4) and PBS (7.4) were used.

Figure 2: Stability of heparin-loaded CS-HA nanosystems in different media: UFH-loaded nanoparticles (▲) and LMWH-loaded nanoparticles (×) in PBS (pH 7.4); UFH-loaded nanoparticles (■) and LMWH-loaded nanoparticles (□) in HBSS (pH.7.4) (mean ± S.D., n=3).

The contact between the nanoparticle formulations with both media

produced an increase in size (relative to the corresponding sizes in water, see

Table 1). This increase may be related to swelling, attributed to the

combination of electrostatic intermolecular repulsion of TPP, HA and heparin

(CS chains are partially uncharged at pH 7.4 and the interactions with

polyanions become weaker) and the ionic strength of these media, as

demonstrated by Lopez-Leon et al. (2005).

The stability of the nanoparticles was also assayed in water at 4°C,

and the nanosystems were stable for up to three months (data not shown).

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107

Heparin release studies from CS-HA nanoparticles

Taking into account the stability profiles of the selected systems, the

release studies were performed in PBS (pH 7.4). The release kinetics of the

different heparins was quite different (Fig. 3). In the case of the systems

loaded with UFH, the drug was released very slowly, with final release of

approximately 10% of the drug within 12 hours of incubation. Otherwise, the

LMWH was released in a faster, continuous manner, with a final release of

approximately 80% in the same period. Considering the high net negative

charge of heparin and the positive charge of CS, a strong electrostatic

interaction between these oppositely charged macromolecules is expected,

resulting in slow drug release. The faster release rate observed for LMWH

may be attributed to its smaller molecular weight, which allows better

diffusion of the drug from the nanoparticles. There was also an appreciable

difference between the drug loading values of the selected systems containing

UFH and LMWH (33.6 and 60.6%, respectively). These different values may

also affect the release profiles obtained for the two systems.

Figure 3: Heparin release from UFH-loaded CS-HA nanoparticles (□) and LMWH-loaded CS-HA nanoparticles (■) in PBS (pH 7.4) (means ± S.D., n=3).


108

Confocal microscopy study of interaction between heparin-loaded CS-

HA nanoparticles and rat mast cells

Taking into account that heparin prevents mast cell degranulation via

an intracellular receptor (Ahmed et al., 2000; Wong and Koh, 2000; Niven

and Argyros, 2003), we aimed to establish whether inclusion of the drug in

CS-HA nanoparticles provides intracellular access to the mast cells. Thus, the

interaction of fluorescent UFH-loaded CS-HA nanoparticles was observed by

confocal microscopy. The overlapping of the fluorescent signal from the

incubated nanoparticles (green) with that corresponding to the mast cells

(red), resulted in an orange colour (Fig. 4.2). This means that the fluorescent

nanoparticles effectively interacted with the mast cells after a period of

contact of two hours. We confirmed that this interaction enables the

nanoparticles to be internalized in mast cells by observing the fluorescent

nanoparticles in sequential slides from the “z” axis of mast cells (Fig. 4.3).

The positive control (Fig. 4.1) indicates that fluorescent mastocytes did not

emit the signal of fluorescent nanoparticles, thus validating the results

obtained.

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Figure 4: Confocal laser scanning microscopy images of fluorescent mastocytes and fluorescent UFH-loaded CS-HA nanoparticles. (1) Mastocytes not incubated with nanoparticles (positive control): (a) excitation signal for mastocytes (red); (b) excitation signal for nanoparticles (no signal); (c) overlapping of both signals (red), and (d) optical signal. (2) Mastocytes after incubation with nanoparticles: (a) excitation signal for mastocytes (red); (b) excitation signal for nanoparticles (green); (c) overlapping of both signals (orange), and (d) optical signal. (3) Slides of mastocytes taken every 1.5 microns in the “z” axis, after incubation with nanoparticles. First line: excitation signal for mastocytes (red); second line: excitation signal for nanoparticles (green).

We also tested LMWH-loaded CS-HA nanoparticles, with similar

results, but finally selected the systems containing UFH as that they are

larger than the former and therefore may be less well internalized by rat mast

cells.


110

Viability test of mast cells after extraction from rats and contact with

UFH or LMWH-loaded nanoparticles.

The viability of rat mast cells was higher than 90% in both cases, as

assessed by trypan blue staining (see Materials and Methods). The selected

dose of UFH or LMWH-loaded nanoparticles to be tested in mast cells

corresponded to the highest dose to be administered in the subsequent

histamine release studies (equivalent to 200 μg/mL of heparin).

Ex vivo studies with rat mast cells: Inhibition of histamine release by

heparin-loaded CS-HA nanoparticles

Considering that heparin-loaded CS-HA nanoparticles were

effectively internalized by the mast cells, we decide to evaluate and compare

the capacity of heparin -administered in the form of solution or loaded in the

selected nanoparticles- to prevent histamine release in rat mast cells. To our

knowledge, this is the first time that such experiments have been carried out

and reported.

Firstly, we tested the effect of different doses of UFH or LMWH in

solution on histamine release by rat mast cells; the cells were previously

stimulated with a standard substance that elicits degranulation by binding to

mast cell granules (compound 48/80) (Ortner and Chignell, 1981). A dose-

dependent effect was found, with no significant differences between the

different types of heparin (Figure 5). This confirmed that, under these

conditions, heparin was effective at preventing histamine release (IC50

(μg/mL) = 6.8±1.2 for UFH and 12.3±3.1 for LMWH), and demonstrated the

effective range of concentrations required for this effect.

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111

Figure 5: Effect of UFH and LMWH solutions on histamine-release from rat mast cells. Histamine release was initiated by incubating the cells with a 100 µM solution of compound 48/80 (black bar), and preincubating different concentrations of UFH (vertical-line bars) or LMWH (horizontal-line bars) before the addition of compound 48/80 (n=3, p˂0.05).

The effect of the UFH-loaded CS-HA nanoparticles in preventing

histamine release, compared with that obtained with UFH solution is shown

in Fig. 6a. The dose-dependent effect was maintained, with no significant

differences between the UFH solution and the UFH-loaded CS-HA

nanoparticles (IC50 (μg/mL) = 6.9±1.4 and 3.6±1.8, respectively). Blank

nanoparticles did not have any effect on the release of histamine from

mastocytes (the concentration of the tested blank nanoparticles was chosen

according to the highest dose of heparin-loaded nanoparticles tested, thus

equivalent to 200 μg/mL of heparin). A similar finding was observed on

comparison of the results obtained with the LMWH in solution and LMWH

encapsulated in the nanoparticles (IC50 (μg/mL) = 5.5±2.1 and 9.6±2.3,

respectively) (Figure 6b).


112

Figure 6: Effect of heparin solutions and heparin-loaded CS-HA nanoparticles on histamine release from rat mast cells. Histamine release was initiated by incubating the cells with a 100 µM solution of compound 48/80 (black bar) and preincubating different concentrations of (a) UFH solution (vertical-line bars), UFH-loaded CS-HA nanoparticles (horizontal-line bars) or (b) LMWH solution (vertical-line bars), LMWH-loaded CS-HA nanoparticles (horizontal-line bars), before the addition of compound 48/80. As a control, the cells were preincubated with a fixed concentration of blank CS-HA nanoparticles (squared bars) before the addition of compound 48/80 (n=3, p<0.05).

The results obtained with heparin-loaded nanoparticles are not so

promising if they are compared with those obtained with heparin solutions.

However, the experimental conditions do not reflect physiological barriers in

airways such as mucociliary clearance (via the mucociliary escalator) and

enzymatic activity. These barriers may be better overcome by the described

b

a

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113

polysaccharide nanosystems because of the mucoadhesive-properties of CS

(Aspden et al., 1997; Lim et al., 2000) and HA (Prichtard et al., 1996; Lim et

al., 2000) and because of the intrinsic capacity of nanoparticles to protect the

loaded drug from the enzymatic attack. Additionally, the nanoparticulate

formulations may improve the effect of a conventional heparin formulation

because of slow drug release, thus prolonging the antiasthmatic effect.

Unfortunately, the experimental ex-vivo conditions do not allow long-term

experiments to be carried out.

Whether CS-HA nanoparticles loaded with heparin can really

improve the effect of heparin in preventing mast cell degranulation can only

be answered by conducting in-vivo experiments. This is the next challenge in

validating our hypothesis.

Conclusions

Nanosystems were produced from CS and HA and their suitability as

heparin carriers for the treatment of asthma was investigated. Confocal

microscopy revealed that heparin-loaded CS-HA nanoparticles were

internalized by rat mast cells. However, the capacity of free heparin and of

heparin encapsulated in the nanosystems to prevent histamine release was

very similar, and showed the same dose-response dependence.

Acknowledgements

The authors acknowledge financial support from the Spanish

Government (SAF 2004-08319-C02-01 and Consolider-Ingenio CSD 2006-

00012); Felipe Oyarzun-Ampuero was in receipt of a CONICYT scholarship.

J.B. received financial support from the Programa Isabel Barreto (Xunta de

Galicia). We also thank Mr. Salvador Arines for technical assistance with the

mast cells assays.


114

References

1. Ahmed, T., Ungo, J., Zhou, M., Campo, C., 2000. Inhibition of allergic

late airway responses by inhaled heparin-derived oligosaccharides. J.

Appl. Physiol., 88, 1721-1729.

2. Aspden, T.J., Mason, J.D., Jones, N.S., Lowe, J., Skaugrud, O., Illum,

L., 1997. Chitosan as a nasal delivery system: the effect of chitosan

solutions on in vitro and in vivo mucociliary transport rates in human

turbinates and volunteers. J. Pharm. Sci. 86(4), 509-13.

3. Buceta, M., Dominguez, E., Castro, M., Brea, J., Alvarez, D., Barcala,

J., Valdes, L., Alvarez-Calderon, P., Dominguez, F., Vidal, B., Diaz,

J.L., Miralpeix, M., Beleta, J., Cadavid, M.I., Loza, M.I., 2008. A new

chemical tool (C0036E08) supports the role of adenosine A(2B)

receptors in mediating human mast cell activation. Biochem.

Pharmacol., 76(7), 912-21.

4. Calvo, P., Remuñan-Lopez, C., Vila-Jato, J.L., Alonso, M.J., 1997.

Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein

carriers. J. Appl. Pol. Sci., 63, 125-132.

5. Campo, C., Molinari, J.F., Ungo, J., Ahmed, T., 1999. Molecular-

weight-dependent effects of nonanticoagulant heparins on allergic

airway responses. J. Appl. Physiol., 86(2), 549-557.

6. De Campos, A.M., Diebold, Y., Carvalho, E.L., Sanchez, A., Alonso,

M.J., 2004. Chitosan nanoparticles as new ocular drug delivery systems:

in vitro stability, in vivo fate, and cellular toxicity. Pharm. Res., 21(5),

803-10.

7. De la Fuente, M., Seijo, B., Alonso, M.J., 2008a. Bioadhesive

hyaluronan-chitosan nanoparticles can transport genes across the ocular

mucosa and transfect ocular tissue. Gene Ther., 15(9), 668-76.

Capitulo 2

115

8. De la Fuente, M., Seijo, B., Alonso, M.J., 2008b. Novel hyaluronan-

based nanocarriers for transmucosal delivery of macromolecules.

Macromol. Biosci., 8(5), 441-50.

9. Garcia-Fuentes, M., Prego, C., Torres, D., Alonso, M.J., 2005. A

comparative study of the potential of solid triglyceride nanostructures

coated with chitosan or poly(ethylene glycol) as carriers for oral

calcitonin delivery. Eur. J. Pharm. Sci., 25(1), 133-43.

10. Green, W.F., Konnaris, K., Woolcock, A.J., 1993. Effect of salbutamol,

fenoterol, and sodium cromoglicate on the release of heparin from

sensitized human lung fragments challenged with Dermatophagoides

pteronyssinus allergen. Am. J. Respir. Cell Mol. Biol., 8, 518-521.

11. Halayko, A.J., Recto, E., Sthepens, N.L., 1997. Characterization of

molecular determinants of smooth muscle cells heterogeneity. Can. J.

Physiol. Pharmacol., 75, 917-919.

12. Johnson, P.R., Armour, C.L., Carey, D., Black, J.L., 1995. Heparin and

PGE2 inhibit DNA synthesis in human airway smooth muscle cells in

culture. Am. J. Physiol., 269, L514-L519.

13. Kanabar, V., Hirst, S.J., O´Connor, B.J., Page, C.P., 2005. Some

structural determinants of the antiproproliferative effect of heparin-like

molecules on human airway smooth muscle. Br. J. Pharmacol., 146,

370-377.

14. Kilfeather, S.A., Tagoe, S., Perez, A.C., Okona-Mensa, K., Matin, R.,

Page, C.P., 1995. Inhibition of serum-induced proliferation of bovine

tracheal smooth muscle cells in culture by heparin and related

glycosaminoglycans. Br. J. Pharmacol., 114, 1442-1446.

15. Köping-Höggård, M., Sanchez, A., Alonso, M.J., 2005. Nanoparticles as

carriers for nasal vaccine delivery. Expert Rev. Vaccines., 4(2),185-96.

16. Lago, J., Alfonso, A., Vieytes, M.R., Botana, L.M., 201. Ouabain-

induced enhancement of rat mast cells response. Modulation by protein

phosphorylation and intracellular pH. Cell Signal., 13(7), 515-24.


116

18. Lim, S.T., Martin, G.P., Berry, D.J., Brown, M.B., 2000. Preparation

and evaluation of the in vitro drug release properties and mucoadhesion

of novel microspheres of hyaluronic acid and chitosan. J. Control. Rel.,

66, 281-292.

18. Lopez-Leon, T., Carvalho, E.L., Seijo, B., Ortega-Vinuesa, J.L., Bastos-

Gonzalez, D., 2005. Physicochemical characterization of chitosan

nanoparticles: electrokinetic and stability behavior. J. Colloid Interface

Sci., 283(2), 344-51.

19. Martinez-Salas, J., Mendelssohn, R., Abraham, W.M., Hsiao, B.,

Ahmed, T., 1998. Inhibition of allergic airway responses by inhaled

low-molecular-weight heparins: molecular-weight dependence. J. Appl.

Physiol., 84(1), 222-228.

20. Molinari, J.F., Campo, C., Shahida, S., Ahmed, T., 1998. Inhibition of

antigen-induced airway hyperresponsiveness by ultralow molecular-

weight heparin. Am. J. Respir. Crit. Care Med., 157, 887-893.

21. Niven, A.S., Argyros, G., 2003. Alternate treatments in asthma. Chest.,

123(4), 1254-65.

22. Ortner, M.J., Chignell, C.F., 1981. The effect of concentration on the

binding of compound 48/80 to rat mast cells: a fluorescence microscopy

study. Immunopharmacology, 3(3), 187-91.

23. Page, C.P., 1991. One explanation of the asthma paradox: inhibition of

natural anti-inflammatory mechanism by beta 2-agonist. Lancet, 337,

717-720.

24. Page, S., Ammit, A.J., Black, J.L., Armour, C.L., 2001. Human mast

cell and airway smooth muscle cell interactions: implications for

asthma. Am. J. Physiol. Lung Cell. Mol. Physiol., 281, L1313-L1323.

25. Prego, C., García, M., Torres, D., Alonso, M.J., 2005. Transmucosal

macromolecular drug delivery. J. Control Release., 101(1-3), 151-62.

26. Prichtard, K., Lansley, A.B., Martin, G.P., Helliwell, M., Marriot, C.,

Benedetti, L.M., 1996. Evaluation of the bioadhesive properties of

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hyaluronan derivates: detachment weight and mucocilliary transport

studies. Int. J. Pharm., 129, 137-145.

27. Robinson, D.S., 2004. The role of the mast cell in asthma: induction of

airway hyperresponsiveness by interaction with smooth muscle? J.

Allergy Clin. Immunol., 114, 58-65.

28. Wong, W.S., Koh, D.S., 2000. Advances in immunopharmacology of

asthma. Biochem Pharmacol., 59(11), 1323-35.

Capitulo 3

A potential nanomedicine consisting in heparin-loaded polysaccharide

nanocarriers for the treatment of asthma

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121

Abstract

The aim of this study is to produce and characterize a new

nanomedicine consisting of chitosan (CS)/carboxymethyl-β-cyclodextrin

(CMβCD) loaded with unfractioned or low-molecular-weight heparin (UFH

or LMWH, respectively), and evaluate its potential in asthma treatment. The

nanoparticles are prepared by ionotropic gelation showing a size ranged

between 221 and 729 nm with a positive zeta potential. The drug association

efficiency is higher than 70%. Developed nanosystems are stable in Hank's

balanced salt solution pH 6.4, releasing slowly the drug. Ex vivo assays,

show that nanocarriers led to an improvement of heparin at preventing mast

cell degranulation. These results agree with the effective cellular

internalization of the fluorescently-labelled nanocarriers, and postulate these

nanomedicines as promising formulations for asthma treatment.

Keywords: chitosan; cyclodextrins; heparin delivery; mast cells;

nanoparticles.

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Introduction

Over the last few decades, the design of new delivery approaches for

the administration of drugs by the pulmonary route has received increasing

attention. These delivery efforts have taken advantage of the physiological

characteristics of this organ (i.e. large superficial area, thin epithelial barrier,

great vascularization, and low proteolitic activity) which offer possibilities

for systemic and local treatments.[1] Within this context, the use of polymeric

carriers represents an attractive strategy for the pulmonary delivery of

macromolecular compounds.[1-3]

A variety of nanocarriers have been investigated for the delivery of

macromolecules to the respiratory tract.[4-10] Among these, chitosan (CS)-

based nanocarriers have shown a degree of success for the delivery of

macromolecules across the nasal and pulmonary mucosae.[11-18] Overall, these

reports have shown that CS-based nanocarriers have an important capacity

for the association of macromolecular drugs and are able to facilitate their

intracellular access, thus leading to a significant enhancement of their in vivo

efficacy.

In our view, a particularly promising CS-based nanocarrier for

pulmonary drug delivery is the one composed of CS and cyclodextrins.

Indeed, using the CALU-3 model epithelial cell line, we have shown that

these hybrid nanoparticles are able to enter in the intracellular space.

Moreover, we could confirm this ability to overcome the epithelial barrier in

vivo following nasal administration.[19-20] These facts together with the

reduction of the cellular toxicity of the nanostructures due to the presence of

cyclodextrins suggest the potential of this nanocarrier for nasal and

pulmonary drug delivery.

Taking into account the favorable characteristics of CS-cyclodextrins

nanoparticles for pulmonary drug delivery, we selected heparin as a drug

which could potentially benefit from this delivery approach. Heparin is a

A potential nanomedicine consisting in heparin-loaded polysaccharide nanocarriers for the treatment of asthma

124

macromolecular drug that has been classically used as an anti-coagulant,

however, recently, there has been an accumulated evidence of the

antiasthmatic activity of this molecule. In particular, the activity of inhaled

heparin has been systematically studied by Ahmed and co-workers in a sheep

model,[21-23] and also in humans.[24-26] This previous work has led to a number

of conclusions such as i) the antiashmatic activity of heparin is related to its

capacity for inhibiting the degranulation of mast cells by interacting with the

intracellular receptor of inositoltrisphosphate (IP3); (ii) the potency of heparin

is inversely proportional to its molecular weight; iii) the antiasthmatic effect

of heparin is independent to its antiacoagulant activity.

Therefore, our aim in this work was to explore the potential of CS-

cyclodextrin nanoparticles for the targeted intracellular delivery of heparin

into mast cells. Therefore, we associated heparin to the nanoparticles and

evaluated their capacity to enter mast cells and preventing histamine release.

Experimental Section

Materials

Ultrapure CS hydrochloride salt (CS; UP CL 113, molecular weight

~125 kDa and degree of acetylation 14%) was purchased from Pronova

Biopolymer AS (Oslo, Norway). Na-carboxymethyl-β-cyclodextrin

(CMβCD, molecular weight 1375 Da) having a substitution degree of 3.0-3.5

was purchased from Fluka GmbH (Buchs, Switzerland). Unfractionated

heparin sodium salt (UFH, molecular weight ~18 kDa, 202 USP units/mg),

low-molecular-weight heparin sodium salt (LMWH, molecular weight ~4

kDa, 53 USP units/mg) and pentasodiumtripolyphosphate (TPP) were

purchased from Sigma Aldrich (Madrid, Spain). All other solvents and

chemicals were of the highest commercially available grade.

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125

Preparation of Nanoparticles

CS-CMβCD nanoparticles loaded with heparin were prepared

according to the procedure previously developed by our group.[27]

Nanoparticles were spontaneously obtained by ionotropic gelation between

the positively charged amino groups of CS and the negatively charged

CMβCD, TPP and heparin. Briefly, 1.5 mL of a mixture of an aqueous

solutions of CMβCD (0-1.33 mg/mL), TPP (0-0.23 mg/mL) and UFH (0.67-

1.4 mg/mL) or LMWH (1.07 mg/mL) were added to 3 mL of a CS solution

(0.2% w/v, pH 4.9) under magnetic stirring at room temperature. Magnetic

stirring was maintained for 10 min to enable complete stabilization of the

system. The nanoparticles were transferred to Eppendorf tubes and isolated

by centrifugation in 20 μL of a glycerol bed (16000 × g, 30 min, 25º C).

Supernatants were collected and the nanoparticles were then suspended in

ultrapure water by shaking on a vortex mixer.

The production yield of the systems was obtained by centrifugation

of fixed volumes of the nanoparticle suspension (16000 × g, 30 min, 25º C),

without the glycerol bed. The supernatants were discarded and the systems

were freeze-dried. The yield was calculated as follows:

100Components theofAmount Total

WeightlesNanopartic Yield ×= (1)

Physicochemical Characterization of Heparin-Loaded CS-CMβCD

Nanoparticles






126

Morphological examination of the nanoparticles was performed by

transmission electron microscopy (TEM) (CM12 Phillips, Netherlands). The

samples were stained with 1% (w/v) phosphotungstic acid for 10 s,

immobilized on copper grids with Formvar® and dried overnight for viewing

by TEM.

Association Efficiency and Drug Loading of Heparin-Loaded CS-

CMβCD Nanoparticles

The association efficiencies of the selected formulations were

determined after isolation of nanoparticles by centrifugation as described in

Section 2.2. The amount of unbound heparin in the supernatant was

determined by a colorimetric method (Stachrom® Heparin, DiagnosticaStago,

France).

The association efficiency of heparin and the drug loading were calculated as

follows:

100drug ofamount Total

drug unbound ofAmount - drug ofamount Total efficiencyn Associatio ×=(2)

100 weightlesNanopartic

drug unbound ofAmount - drug ofamount TotalLoading Drug ×=(3)

Stability Study

Selected nanoparticles formulations were prepared and centrifuged in

the presence of glycerol. Nanoparticles were tested for their stability taking

into account the change in size of nanoparticles and possible precipitations in

different media at 37º C, including: Hank's balanced salt solution (HBSS) at

pH 6.4 and 7.4, and phosphate buffered saline (PBS), at pH 7.4 (for

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127

composition of these solutions, see below). Nanoparticles were incubated in

these media and samples were collected at several time intervals (0, 1, 3, 5,

10 and 24 h), and the size distribution of the nanoparticles was measured by

photon correlation spectroscopy.

The composition of HBSS was: 137 mMNaCl, 5.4 mMKCl, 0.25 mM

Na2HPO4, 0.44 mM KH2PO4 and 4.2 mM NaHCO3.

The composition of PBS was: 137 mMNaCl, 2.7 mMKCl, 1.4 mM

NaH2PO4 and 1.3 mM Na2HPO4.

In Vitro Heparin Release Studies from CS-CMβCD Nanoparticles

Heparin release studies were performed by incubating 0.1 mg of the

selected nanoparticles in 1 mL of HBSS (pH 6.4) at 37º C. The sample were

centrifuged at appropriate time intervals (1, 5 and 12 h), and the amount of

heparin released was evaluated with the heparin kit described above.

Study of Interaction of Fluorescent Heparin-Loaded CS-CMβCD

Nanoparticles with Rat Mast Cells by Confocal Microscopy

Fluorescein Labelling to CS: CS was labelled with fluorescein following a

slight modification of the method described by De Campos et al.[28] The

covalent attachment of fluorescein to CS was by the formation of amide

bonds between primary amino groups of the polymer and the carboxylic acid

groups of fluorescein. Briefly, 250 mg of CS was dissolved in 25 mL of

water, and 10 mg of fluorescein (Sigma Aldrich, Spain) was dissolved in 1

mL of ethanol. These solutions were then mixed, and EDAC (1-ethyl-3-

(dimethylaminopropyl) carbodiimide hydrochloride) (Sigma Aldrich, Spain)

was added to a final concentration of 0.05 M, to catalyze the formation of

amide bonds. The reactive mixture was incubated under permanent magnetic

stirring for 12 h in the dark, at room temperature. The resulting conjugate


128

was finally isolated by dialysis for 72 h (cellulose dialysis tubing, pore size

12400 Da; Sigma Aldrich, Spain) against demineralised water, and freeze-

dried. The pH of fluorescent CS was adjusted to the same value as the raw

CS solution (pH 4.9) with HCl, for the preparation of fluorescent

nanoparticles.

Preparation of Fluorescent Heparin-Loaded CS-CMβCD Nanoparticles:

Fluorescent nanoparticles were prepared according to the same procedure

explained in 2.2. The selected mass distribution for the preparation of

fluorescent nanoparticles was: 6 mg of fluorescent CS, 0.85 mg of CMβCD,

0.34 mg of TPP and 1.6 mg of UFH.

Confocal Laser Scanning Microscopy Study: An aqueous solution (50 μL)

containing 0.3 mg of isolated fluorescent UFH-loaded CS-CMβCD

nanoparticles was incubated with 450 μL of a suspension of mast cells

(10x103 cells/100 μL) in Umbreit (for composition, see below) containing

0.05% w/v of BSA. The mixture was incubated for 2 h at 37 °C, and the cells

were then separated by centrifugation (10 min, 200xg) and discarding the

supernatants. Two hundred μL of Umbreit+BSA solution (at 4 °C) were then

added to the cell pellet. The pellet was resuspended and centrifuged again to

extract the non-internalized nanoparticles. This procedure was repeated once

more. Mast cells were fixed for 5 minutes in paraformaldehyde (2% w/v, 100

μL) and washed 3 times with the Umbreit+BSA solution, by centrifugation.

Two hundred μL of a Bodipi® phalloidin solution (Invitrogen, USA) were

added to the cell pellet and the cells were incubated for 30 min at room

temperature. The cells were washed 3 times (Umbreit+BSA) by

centrifugation, the supernatant was discarded, and the pellet was resuspended

in 20 μL of the Umbreit+BSA solution. The resuspended sample was placed

on the surface of a positively charged microscope slide (Superfrost Ultra

Plus, Menzel-Glaser, Irland) and dried at room temperature overnight. The

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sample was prepared in Vectashield medium (Vector, USA) for visualization

by confocal microscopy (CLSM, Zeiss 501, Germany) (all of the described

procedures were carried out in darkness to prevent the loss of the fluorescent

signal from the nanoparticles and mast cells).

The composition of Umbreit saline solution was: 1.2 mM MgSO4, 1.2 mM

NaPO4H2, 22.85 mM NaHCO3, 5.94 mMKCl, 1 mM CaCl2, 119 mMNaCl

and 0.1% glucose.

Ex Vivo Studies With Rat Mast Cells: Inhibition of Histamine Release by

Heparin-Loaded CS-CMβCD Nanoparticles

Animal procedures were conducted in accordance with the standard

ethical guidelines (National Institutes of Health, 1995; Council of Europe,

1996) and approved by the local ethical committees.

Rat Mast Cell Purification and Viability: Mast cells were obtained by lavage

of pleural and peritoneal cavities of female Sprague - Dawley rats (400–800

g) with Umbreit saline solution, following procedures similar to those

described in other studies.[29-30] The suspension obtained from each rat was

centrifuged at 100xg for 5 min (4 ºC) and suspended in a final volume of 1

mL of Umbreit containing 0.05% w/v of BSA. Purification was carried by

centrifugation on 4 mL of an isotonic Percoll gradient at 600 g for 10 min (4

ºC). The mast cells were washed twice with the Umbreit+BSA solution and

maintained at 4 °C in this solution until use. Mast cells were quantified by

toluidin blue staining (95% purity) and the viability (90%) assessed by trypan

blue staining (the procedure is described below).


130

Trypan blue staining procedure: Mast cell viability studies were carried out

by trypan blue staining in an inverted microscope, as described by Lago et

al.[29] This involved visual counting of the stained cells in the five fields of a

counting chamber. The percentage of viability was calculated with the

following formula:

100CellsMast ofNumber Total

Fields Five theFromMean Aritmetic Cells Stained BlueTrypan ×=(4)

In order to test the mast cell viability after contact with heparin-

loaded nanoparticles, the same procedure was used, and the UFH or LMWH-

loaded CS-CMβCD nanoparticles added to the rat mast cell suspension

(1x105 cells per test tube). The tested dose of nanoparticles was equivalent to

200 μg/mL of UFH or LMWH.

Measurement of Histamine Release in Rat Mast Cells: Rat mast cells (1x105

cells per test tube) were pre-warmed at 37 ºC (10 min) in BSA-free Umbreit

saline solution containing the UFH or LMWH solutions or the nanoparticles

loaded with UFH or LMWH. Histamine release from mast cells was then

initiated by incubating the cells with 100 µM of compound 48/80 (Sigma

Aldrich, Spain) for 20 min at 37 ºC. The cells were then centrifuged at

1100xg for 3 min at 4 ºC, and two aliquots (100 µl) of the supernatants were

collected in a 96-well microplate. The rest of the supernatants were discarded

and the pellets were resuspended in 500 μL of HCl 0.1 M, sonicated for 1

min and centrifuged at 1100 xg for 6 min. Two aliquots of 100 μL of the

supernatants were collected for residual histamine determination. Histamine

was assayed fluorometrically, as described by Lago et al.[29]; briefly, 80 µL

NaOH 1 M were added to 100 µL of the sample, then 50 µl phthaldialdehyde

0.04% w/v were added to each well and plate was incubated for 4 min at 25°

C. After this time, 50 µl of 3 M HCl were added and fluorescence was

measured within 20 min, at excitation and emission wavelengths of 360 nm

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131

and 465 nm respectively, in a Tecan Ultra Evolution reader (Tecan,

Switzerland).

Data Analysis for Measurement of Histamine Release in Rat Mast Cells:

Results were expressed as percentage of the total histamine released after

stimulation with compound 48/80. The results were corrected for

spontaneous histamine release in the absence of any chemical and under the

same conditions. The equation used for the calculation was

HR=[(S−ER)/(S+P−ER)]×100, where HR is the percentage histamine

release; S, supernatant fluorescence; ER, fluorescence of spontaneous release

supernatants and P, pellet fluorescence.

IC50 values were obtained by fitting the data with non-linear

regression, with Prism 2.1 software (GraphPad, San Diego, CA).

Statistical Analysis

The statistical significance of the differences between formulations

was determined by application of two-way analysis of variance (ANOVA)

followed by a two-tailed paired Student’s test. Differences were considered

significant at p<0.05.

Results and Discussion

It has been reported that heparin could potentially be used for the

treatment of asthma. However, this potential use is constrained by its limited

access into the mast cells where target receptors for preventing degranulation

are located.[31-32] This limited access could be related to the electrostatic

repulsion between this highly negative macromolecule and the mastocyte

membranes. Therefore, the hypothesis of this work was that the incorporation


132

of heparin into nanocarriers could neutralize its charge and facilitate the

internalization and controlled release of the drug into the mast cells.

Preparation and Characterization of Heparin-Loaded CS-CMβCD

Nanoparticles

Nanoparticles loaded with heparin were prepared by the ionotropic

gelation technique. The ability of CS to form a gel after contact with

polyanions by promoting inter and intramolecular linkages enables the

formation of the nanoparticles.[33] In this case, an ionic interaction occurs

between the positively charged CS and the negatively charged CMβCD,

heparin and the polyanion TPP. The ionic gelation process is extremely

simple and involves mixing two aqueous phases at room temperature.

As a first approximation for the formation of adequate nanoparticle

formulations, we assayed different ratios of the three anionic components of

the nanomedicines. Then, we identified the ratio between components that

enabled the formation and also the adequate isolation of nanosystems. Table

1 shows size, polydispersity index, and zeta potential of a variety of

formulations tested with UFH. Valuable information extracted from these

experiments is that when the amount of polyanions was too low (relative to

CS), nanoparticles could not be formed. However, if the amount of

polyanions was too high, the particles precipitated or aggregated during the

isolation process. This behavior could be attributed to the gradual

counterionization of the positively charged CS, as noted by the reduction in

the positive zeta potential values of the nanosystems.

All the resulting nanosystems loaded with UFH exhibited a size in

the range of 350-730 nm; polydispersity values were between 0.2-1 and the

positive zeta potential ranged from +33.2 to +40.7 (Table 1). These ranges of

values are similar to those previously presented by Krauland et. al.[27] who

developed different formulations of UFH loaded in CS-CMβCD or CS-TPP

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nanoparticles. Among the formulation developed in this study, the one

comprising 6 mg of CS, 0.85 mg of CMβCD, 0.34 mg of TPP and 1.6 mg of

UFH was selected for further studies including in vitro characterization and

ex vivo efficacy.

Table 1: Physicochemical properties of the nanoparticles prepared with different ratios of CS-CMβCD-TPP-heparin (mean ± S.D., n=3).

Amount [mg] CS-CMβCD-TPP-

heparin

Size [nm]

Polydispersity Index

Zeta potential [mV]

6-1-0-1a 359 ± 21 0.3 – 0.4 +40.7 ± 1.0 6-2-0-2a 729 ± 54 0.8 – 1 +33.2 ± 0.8

6-2-0-2.1a Non resuspendable --- --- 6-0.85-0.34-1.6a 375±69 0.3 – 0.5 +37.0 ± 1.6

6-1-0.34-1.6a 473 ± 24 0.6 – 0.9 +34.7 ± 1.2 6-1.15-0.34-1.6a Non resuspendable --- --- 6-1.3-0.34-1.6a Precipitation --- ---

6-0.85-0.34-1.6b 221± 26 0.2 – 0.3 +36.8 ± 0.7 a= UFH; b = LMWH

Importantly, the association efficiency of UFH in the selected formulation

was 77.0%. This high association value is related to the capacity of the

polyanion to interact avidly with the polycationic CS as previously shown for

other CS-based nanosystems.[27,30]

With the aim of elucidating the influence of the heparin Mw in the in

vitro and ex vivo behavior of the heparin-loaded nanoparticles, we also used

a low-molecular-weight-heparin (LMWH). This new formulation exhibited

similar values of zeta potential and association efficiency, however the size

and polydispersity values were smaller when compared to those of high Mw

heparin-loaded nanoparticles (Table 1). This could be attributed to a tighter

assembling of the components forming the nanoparticles. The differences

between the two formulations are also illustrated in the TEM micrographs

presented in Figure 1a and b. It should be noted that the nanosystems

containing LMWH form a smaller and denser structure than those containing


134

UFH. The photographs also show the spherical shape of nanosystems, this is

in agreement with previous works describing nanoparticles prepared

following the same method.[27,30]

Figure 1: Electron transmission micrographs of CS-CMβCD nanoparticles containing UFH (a) or LMWH (b).

Stability Studies

The stability of the selected systems was investigated in media that

mimics biological conditions and that are usually used in cell culture studies.

These media included: HBSS (pH 6.4 and 7.4) and PBS (pH 7.4). The

stability of the selected nanoparticles loaded with UFH and LMWH was

maintained for up to 24 h in HBSS pH 6.4 (Figure 2) while in the other media

the nanoparticles aggregated immediately (data not shown). The zeta values

of the selected UFH/LMWH-loaded nanoparticles in HBSS pH 6.4 were

approximately +14 mV, due to the presence of ionizable groups of CS in this

medium. In contrast, the zeta values of the systems in HBSS pH 7.4 and PBS

7.4 were neutral (≈0 mV), thus, making the systems vulnerable to

aggregation. The stability of the nanoparticles was also assayed in water at 4°

C, where formulations maintained stable for up three months (data not

shown).

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135

Figure 2: Stability of heparin-loaded CS-CMβCD nanosystems in HBSS pH 6.4: UFH-loaded nanoparticles (■) and LMWH-loaded nanoparticles (×) (mean ± S.D., n=3).

Release Studies From Selected Nanosystems

As shown in Figure 3, the release kinetics of heparins in HBSS pH

6.4 was slow and clearly dependent on their molecular weight. In the case of

the systems comprising LMWH, the drug was released very slowly during

the first hour (approximately 10% of the encapsulated drug) followed by a

plateau phase with little further change up to 12 h of incubation. Otherwise,

the UFH was released in a faster, continuous manner, with a final release of

approximately 50% in the same period of incubation. The slower release rate

observed for LMWH could be attributed to the more packed structure of

these nanosystems as we previously argument. The slow release rate

observed for nanosystems loaded with UFH or LMWH agrees with the

results obtained for other formulations comprising CS and heparin and is

attributed to the strong ionic interaction among the anionic drug and the

cationic polymer.[30]


136

Figure 3: Heparin release from UFH-loaded CS-CMβCD nanoparticles (□) and LMWH-loaded CS-CMβCD nanoparticles (■) in HBSS pH 6.4 (means ± S.D., n=3).

Study of Interaction of Heparin-Loaded CS-CMβCD Nanoparticles with

Rat Mast Cells by Confocal Microscopy

Taking into account that the antiasthmatic effect of heparin is

attributed to its capacity of preventing the mast cell degranulation via the

intracellular receptor of IP3,[34-35] the capacity of the selected nanosystems to

gain intracellular access is a fundamental requisite. Thus, we performed

confocal microscopy experiments for visualizing the interaction-transport of

fluorescent heparin-loaded CS-CMβCD nanoparticles in rat-mast cells. In

Figure 4.2, it can be seen that the overlapping of the fluorescent signal from

the incubated UFH-loaded CS-CMβCD nanoparticles (green) with that

corresponding to the mast cells (red), resulted in an orange color. This means

that the fluorescent nanoparticles effectively interacted with the mast cells

after a period of contact of two hours. We confirmed that this interaction

enables the nanoparticles to be internalized in mast cells by observing the

fluorescent nanoparticles in sequential slides from the “z” axis of mast cells

(Fig. 4.3). The positive control (Fig. 4.1) indicates that fluorescent

mastocytes did not emit the signal of fluorescent nanoparticles. The confocal

images obtained upon treatment of cells with fluorescent LMWH-loaded CS-

CMβCD nanoparticles, were similar to those reported in Figure 4.3. Overall,

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137

this capacity of heparin-loaded CS-CMβCD nanoparticles for entering in the

rat mast cells is similar to that previously observed for heparin-loaded CS-

hyaluronic acid nanoparticles.[30] This capacity could be attributed to the

nanometric characteristic of the systems and also to the capacity CS for

interacting with cellular membranes and promoting the intracellular access of

the nanosystems.[20, 36]

Figure 4: Confocal laser scanning microscopy images of fluorescent mastocytes and fluorescent UFH-loaded CS-CMβCD nanoparticles. (1) Mastocytes not incubated with nanoparticles (positive control): (a) excitation signal for mastocytes (red); (b) excitation signal for nanoparticles (no signal); (c) overlapping of both signals (red), and (d) optical signal. (2) Mastocytes after incubation with nanoparticles: (a) excitation signal for mastocytes (red); (b) excitation signal for nanoparticles (green); (c) overlapping of both signals (orange), and (d) optical signal. (3) Slides of mastocytes taken every 1.5 microns in the “z” axis, after incubation with nanoparticles. First line: excitation signal for mastocytes (red); second line: excitation signal for nanoparticles (green).


138

Ex Vivo Studies with Mast Cells for Preventing the Histamine Release

With the aim of evaluating the potential of these new prototypes as

nanomedicines for treating asthma, we tested and compared the capacity of

heparin solutions and heparin-loaded CS-CMβCD nanoparticles to prevent

histamine release in rat mast cells. The histamine release was initiated by

stimulating the cells with a standard substance that elicits degranulation by

binding to mast cell granules (compound 48/80).[37] Figure 5a shows the

effect of the formulation containing UFH on histamine release. The results

show a dose-dependent effect for both UFH solution and UFH-loaded

nanoparticles. This behavior is similar to the one previously reported for

UFH-loaded CS-hyaluronic acid nanoparticles.[30] Importantly, a dramatic

effect in terms of preventing histamine release was achieved for the highest

dose of UFH-loaded CS-CMβCD nanoparticles (≈90% of inhibit inhibition);

an effect that was much less important for UFH in solution (≈55% of

inhibition). This positive behavior contrasts with the one reported for CS-AH

nanoparticles, which effect at preventing histamine release was similar than

that obtained with heparin administered in solution.[30] Consequently, we

attributed such positive effect to the presence of CMβCD in the formulation

and its recognized role in improving the permeability of drugs through

cellular membranes. Moreover, the dose-dependent positive effect of

nanoparticles could be related to the dose-dependent permeabilizing effect of

ciclodextrins. Blank nanoparticles did not have any significant effect on the

release of histamine from mastocytes (the concentration of the tested blank

nanoparticles was equivalent to those administered when 200 μg/mL of

heparin was administered in the nanoparticles).

On the other hand, as shown in Figure 5b, the systems containing

LMWH, produce an effect that is similar to those observed for the UFH-

loaded nanosystems. Again, the effect obtained with the highest tested dose

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of heparin-loaded nanoparticles significantly improved those obtained upon

treatment with LMWH in the solution.

Figure 5: Effect of heparin solutions and heparin-loaded CS-CMβCD nanoparticles on histamine release from rat mast cells. Histamine release was initiated by incubating the cells with a 100 µM solution of compound 48/80 and preincubating different concentrations of (a) UFH solution and UFH-loaded CS-CMβCD nanoparticles or (b) LMWH solution and LMWH-loaded CS-CMβCD nanoparticles, before the addition of compound 48/80. As a control, the cells were preincubated with a fixed concentration of blank CS-CMβCD nanoparticles before the addition of compound 48/80 (n=3, p<0.05).

Importantly, the viability of the mast cells after the addition of the

UFH/LMWH-loaded nanoparticles was, in all cases, similar to that presented

a

b

*

*

*


140

by mastocytes after the extraction from rats (90%), thus discarding any toxic

effect of the tested nanoparticles-doses.

The results obtained with heparin-loaded CS-CMβCD nanoparticles

in terms of preventing histamine release are promising and indicate that

nanoparticle-composition is crucial in order to achieve the desired effect.[30]

These results together with those previously reported on the low toxicity of

CS-CMβCD nanoparticles in CALU-3 cells,[20] support the potential use of

these nanoparticles for the treatment of asthma.

Finally, it is important to note that the ex vivo conditions carried out

in these works do not reflect the physiological barriers in the airways such as

mucociliary clearance (via the mucociliary escalator) and enzymatic activity.

These barriers should be overcome by the described polysaccharide

nanosystems because of the mucoadhesive-properties of CS,[38-39] and the

intrinsic capacity of nanoparticles to protect the loaded drug from enzymatic

attack. In fact, there are some examples in the literature showing the topical

efficacy of CS nanoparticles for the pulmonary delivery of active molecules

for treating tuberculosis[40-41] and cancer[42].

In addition, it could be expected that the nanoparticle-formulations

presented here would improve the effect of a conventional heparin

formulation because of the slow drug release. Unfortunately, the

experimental conditions do not allow long-term experiments to be carried out

for validating the above hypothesis. Ultimately, in vivo experiments would

be necessary in order to validate the potential of these new nanomedicines for

the treatment of asthma.

Capitulo 3

141

Conclusion

Nanosystems were produced from CS and CMβCD and their

suitability as heparin carriers for the treatment of asthma was investigated.

Confocal microscopy revealed that heparin-loaded CS-CMβCD nanoparticles

were internalized by rat mast cells. Experiments in mast cells indicated that

the association of heparin to the nanocarriers led to a significant

improvement of the efficacy of this drug measured in terms of preventing

mast cell degranulation. To the best of our knowledge, this is the first

evidence of the enhanced efficacy of heparin presented in the form of a

nanomedicine for the treatment of asthma.

Acknowledgements: The authors acknowledge financial support from the

Spanish Government (SAF 2004-08319-C02-01 and Consolider-Ingenio CSD

2006-00012); Felipe Oyarzun-Ampuero was in receipt of a CONICYT

scholarship. J.B. received financial support from the Programa Isabel Barreto

(Xunta de Galicia). We also thank Mr. Salvador Arines for technical

assistance with the mast cells assays.

References

1. M. Bur, A. Henning, S. Hein, M. Schneider, C.M. Lehr, Inhalation

Toxicology 2009, 21(S1), 137.

2. B.D. Curmi, J. Kayat, V. Gajbhiye, R.K. Tekade, N.K. Jain, Expert

Opin. Drug Deliv. 2010, 7(7), 781.

3. H.H. Mansour, Y.S. Rhee, X. Wu, Int. J. Nanomed. 2009, 4, 299.

4. M.N.V.R. Kumar, S.S. Mohapatra, X. Kong, P.K. Jena, U. Bakowsky,

C.M. Lehr, J. Nanosci. Nanotech. 2004, 48, 990.


142

5. N. Nafee, S. Taetz, M. Schneider, U.F. Schaefer, C.M. Lehr,

Nanomedicine 2007, 3(3), 173.

6. M. Bivas-Benita, S. Romeijn, H.E. Junginger, G. Borchard, Eur. J.

Pharm. Biopharm. 2004a, 58(1), 1.

7. M. Bivas-Benita, R. Zwier, H.E. Junginger, G. Borchard, Eur. J.

Pharm. Biopharm. 2005, 61, 214.

8. M. Bivas-Benita, T.H.M. Ottenhoff, H.E. Junginger, G. Borchard, J.

Control. Rel. 2005, 107, 1.

9. A. Misra, A.J. Hickey, C. Rossi, G. Borchard, H. Terada, K. Makino,

P.B. Fourie, Colombo, P. Tuberculosis, 2011, 91(1), 71.

10. S. Taetz, N. Nafee, J. Beisner, K. Piotrowska, C. Baldes, T.E. Muedter,

H. Huwer, M. Schneider, U.F. Schaefer, U. Klotz, C.M. Lehr, C.M,

Eur. J. Pharm. Biopharm. 2008, 722, 358.

11. M. Köping-Höggård, A. Sanchez, M.J. Alonso, Expert Rev. Vaccines

2005, 4(2), 185.

12. M. Köping-Höggård, M.M. Issa, T. Köhler, A. Tronde, K.M. Vårum,

P. Artursson, J. Gene Med. 2005, 7(9), 1215.

13. M.M. Issa, M. Köping-Höggård, K. Tømmeraas, K.M. Vårum,

B.E. Christensen, B.E, S.P. Strand, P. Artursson, J Control Rel. 2006,

115(1), 103.

14. N. Csaba, M. Köping-Höggård, M.J. Alonso, Int. J. Pharm. 2009,

382(1-2), 205.

15. N. Csaba, M. Garcia-Fuentes, M.J. Alonso, Adv. Drug Deliv.

Rev. 2009, 61(2), 140.

16. N. Csaba, M. Köping-Höggård, E. Fernandez-Megia, R. Novoa-

Carballal, R. Riguera, M.J. Alonso, J. Biomed. Nanotechnol.

2009, 5(2), 162.

17. C. Prego, M. García, D. Torres, M.J. Alonso, J. Control Rel. 2005

101(1-3), 151.

Capitulo 3

143

18. A. Vila, A. Sánchez, K. Janes, I. Behrens, T. Kissel, J.L. Vila-Jato,

M.J. Alonso, Eur. J. Pharm. Biopharm. 2004, 57(1), 123.

19. D. Teijeiro-Osorio, C. Remuñan-Lopez, M.J. Alonso, Biomacromol.

2009, 10(2), 243.

20. D. Teijeiro-Osorio, C. Remuñan-Lopez, M.J. Alonso, Eur. J. Pharm.

Biopharm. 2009, 71(2), 257.

21. T. Ahmed, C. Campo, M.K. Abraham, J.F. Molinari, W.M. Abraham,

D. Ashkin, T. Syriste, L.O. Andersson, C.M. Svahn, Am. J. Respir.

Crit. Care. Med. 1997, 155(6), 1848.

22. J. Martinez-Salas, R. Mendelssohn, W.M. Abraham, B. Hsiao, T.

Ahmed, J. Appl. Physiol. 1998, 84(1), 222.

23. C. Campo, J.F. Molinari, J. Ungo, T. Ahmed, J. Appl. Physiol. 1999,

86(2), 549.

24. T. Ahmed, J. Garrigo, I. Danta, N. Engl. J. Med. 1993, 329(2), 90.

25. T. Ahmed, B.J. Gonzalez, I. Danta, Am. J. Respir. Crit. Care. Med.

1999, 160(2), 576.

26. J. Garrigo, I. Danta, T. Ahmed, Am. J. Respir. Crit. Care. Med. 1996,

153(5), 1702.

27. A.H. Krauland, M.J. Alonso, Int. J. Pharm. 2007, 340, 134.

28. A.M. De Campos, Y. Diebold, E.L. Carvalho, A. Sanchez, M.J.

Alonso, Pharm. Res. 2004, 21(5), 803.

29. J. Lago, A. Alfonso, M.R. Vieytes, L.M. Botana, Cell Signal 2001,

13(7), 515.

30. F.A. Oyarzun-Ampuero, J. Brea, M.I. Loza, D. Torres, M.J. Alonso.

Int. J. Pharm. 2009, 381(2), 122.

31. J. Lucio, J. D'Brot, C.B. Guo, W.M. Abraham, L.M. Lichtenstein,

A. Kagey-Sobotka, T. Ahmed, Appl. Physiol. 1992, 73(3), 1093.


144

32. T. Ahmed, T Syriste, R. Mendelssohn, D. Sorace, E. Mansour, M.

Lansing, W.M. Abraham, M.J. Robinson, J. Appl. Physiol. 1994, 76(2),

893.

33. P. Calvo, C. Remuñan-Lopez, J.L. Vila-Jato, M.J. Alonso, J. Appl. Pol.

Sci. 1997, 63, 125.

34. W.S. Wong, D.S. Koh, Biochem. Pharmacol. 2000, 59(11), 1323.

35. A.S. Niven, G. Argyros, Chest 2003, 123(4), 1254.

36. M. de la Fuente, N. Csaba, M. Garcia-Fuentes, M.J. Alonso,

Nanomedicine (Lond.). 2008, 3(6), 845.

37. M.J. Ortner, C.F. Chignell, Immunopharmacology 1981, 3(3), 187.

38. T.J. Aspden, J.D. Mason, N.S. Jones, J. Lowe, O. Skaugrud, L. Illum,

J. Pharm. Sci. 1997, 86(4), 509.

39. S.T. Lim, G.P. Martin, D.J. Berry, M.B. Brown, J. Control. Rel. 2000,

66, 281.

40. M. Bivas-Benita, K.E. van Meijgaarden, K.L. Franken, H.E. Junginger,

G. Borchard, T.H. Ottenhoff, A. Geluk, Vaccine 2004b, 22(13-14),

1609.

41. Z. Ahmad, S. Sharma, G.K. Khuller, Int. J. Antimicrob. Agents 2005,

26(4), 298.

42. H. Jin, T.H. Kim, S.K. Hwang, S.H. Chang, H.W. Kim, H.K.

Anderson, H.W. Lee, K.H. Lee, N.H. Colburn, H.S. Yang, M.H. Cho,

C.S. Cho, Mol. Cancer Ther. 2006, 5(4), 1041.

Discusión

145

DISCUSIÓN

Como se ha mencionado en la sección de Introducción, la heparina

posee un interesante potencial como tratamiento del asma bronquial. Sin

embargo, este potencial se ve notablemente limitado por su acceso

restringido al interior de los mastocitos, donde se encuentran localizados los

receptores que pueden prevenir la desgranulación de estas células95;96. La

dificultad en el acceso intracelular de la macromolécula se relaciona con la

repulsión electrostática entre la heparina, que posee una gran densidad de

carga negativa, y las membranas celulares de los mastocitos. Por lo tanto, la

incorporación de la heparina en sistemas nanoparticulares se abre como una

posibilidad de ocultar su carga y, con ello, facilitar su internalización,

además de ofrecer la posibilidad de modular su liberación en el interior de los

mastocitos.

Para la elaboración de los nanosistemas, se seleccionó el polisacárido

quitosano, en combinación con el ácido hialurónico (HA), o con el

oligosacárido carboximetil-β-ciclodextrina (CMβCD). La técnica elegida

para la elaboración de los nanosistemas fue la gelificación ionotrópica y las

principales variables de formulación investigadas en la formación de las

nanopartículas, fueron la proporción de los componentes en cada tipo de

sistema y el peso molecular de la heparina.

95 Lucio J, D'Brot J, Guo CB, Abraham WM, Lichtenstein LM, Kagey-Sobotka A, Ahmed T. (1992). Appl. Physiol. 73(3):1093-101. 96 Ahmed T, Syriste T, Mendelssohn R, Sorace D, Mansour E, Lansing M, Abraham WM, Robinson MJ. (1994). J. Appl. Physiol. 76(2):893-901.

Discusión

146

Preparación de las nanopartículas

Las nanopartículas se prepararon de acuerdo con el procedimiento de

gelificación iónica, previamente desarrollado en nuestro grupo de

investigación97. Esta técnica consiste en mezclar dos fases que contienen

disoluciones acuosas con moléculas de carga positiva y negativa (Fig. 1). Las

nanopartículas se obtienen espontáneamente tras la nanogelificación que se

produce por la interacción entre los grupos amino cargados positivamente del

quitosano y las cargas negativas de los polímeros u oligómeros, en presencia

del agente reticulante (tripolifosfato; TPP). Es un procedimiento

extremadamente simple y suave que se realiza a temperatura ambiente.

Figura 1. Esquema de preparación de los nanosistemas mediante el procedimiento de gelificación iónica (CS:quitosano).

97 Calvo P, Remuñan-Lopez C, Vila-Jato JL, Alonso MJ. (1997) J. Appl. Polymer Sci. 63, 125-132.

Discusión

147

Para la preparación de los nanosistemas de quitosano-CMβCD y

quitosano-HA, conteniendo heparina, fue necesario establecer las

proporciones más adecuadas de los componentes que permitían su apropiada

formación y aislamiento. En general, independientemente de la composición

específica de cada sistema, las condiciones de formación se ajustaron a una

pauta similar.

En las Tablas 1a y 1b se puede apreciar que, en la medida en que se

añadieron cantidades crecientes de los polianiones, se observó un aumento de

tamaño en los sistemas junto con una disminución en el valor absoluto del

potencial zeta. Esto es explicable en términos de la contraionización que el

policatión (quitosano) va sufriendo producto de la adición de cantidades

crecientes de polianiones. Cuando los sistemas se vuelven irresuspendibles, o

precipitan, se supone una la completa contraionización del policatión, que

impide a los sistemas tener una carga eléctrica superficial suficientemente

alta que les permita repelerse electrostáticamente y mantener su estabilidad

coloidal98.

Tabla 1. Características físico-químicas de las nanopartículas preparadas usando diferentes proporciones de (1) CS-HA-TPP-heparina y (2) CS-CMβCD-TPP-heparina (media ± d.e.; n=3).

Cantidad (mg) CS-HA-TPP-

heparina

Tamaño (nm)

Índice de polidispersión

Potential Z (mV)

4-1.2-0.21-1.0 a 201 ± 24 0.2 – 0.4 +32.1 ± 1.6 4-1.2-0.21-1.2 a 217 ± 30 0.2 – 0.4 +28.1 ± 0.9 4-1.2-0.21-1.4 a No resuspendible --- --- 4-1.2-0.21-1.6 a Precipitación --- --- 4-0.6-0.21-1.2 a 162 ± 17 0.1 – 0.3 +34.6 ± 0.6 4-0.6-0.21-1.4 a 193 ± 32 0.2 – 0.5 +32.5 ± 1.7 4-0.6-0.21-1.5 a No resuspendible --- --- 4-0.6-0.21-1.4 b 152 ± 10 0.2 – 0.3 +33.0 ±1.3

CS= quitosano; a= UFH; b= LMWH.

98 Krauland AH, Alonso MJ. (2007). Int. J. Pharm. 340:134-42.

1)

Discusión

148

Cantidad (mg) CS-CMβCD-TPP-heparina

Tamaño (nm)

Índice de polidispersión

Potencial Z (mV)

6-1-0-1a 359 ± 21 0.3 – 0.4 +40.7 ± 1.0 6-2-0-2a 729 ± 54 0.8 – 1 +33.2 ± 0.8

6-2-0-2.1a No resuspendible --- --- 6-0.85-0.34-1.6a 375±69 0.3 – 0.5 +37.0 ± 1.6

6-1-0.34-1.6a 473 ± 24 0.6 – 0.9 +34.7 ± 1.2 6-1.15-0.34-1.6a No resuspendible --- --- 6-1.3-0.34-1.6a Precipitación --- ---

6-0.85-0.34-1.6b 221± 26 0.2 – 0.3 +36.8 ± 0.7

Caracterización de las nanopartículas

Como se puede apreciar en la Tabla 1.1, todos los sistemas de

quitosano-HA presentaron un rango de valores de tamaño, polidispersión y

potencial zeta relativamente estrecho (152-217 nm, 0.1-0.5, y 33.2-40.7 mV,

respectivamente). Por otro lado, los sistemas de quitosano-CMβCD (Tabla

1.2) presentaron una mayor dispersión en los citados parámetros (221-729

nm, 0.2-1, y +33-+41 mV), siendo estos valores similares a los presentados

por Krauland y col. (2007) para diferentes formulaciones nanopartículas de

quitosano-TPP y quitosano-CMβCD conteniendo heparina no fraccionada

(UFH)98. En las formulaciones en las que sustituyó la UFH por heparina de

bajo peso molecular (LMWH), los parámetros de tamaño y polidispersión

fueron los más bajos de todos los sistemas ensayados, lo que, evidentemente,

se relaciona con el menor peso molecular del fármaco. En el caso del

potencial zeta, se puede apreciar que, en todas las formulaciones, se obtienen

valores altamente positivos, lo cual es indicativo de que la superficie de los

nanosistemas está compuesta principalmente por quitosano.

Para las siguientes etapas de caracterización y evaluación, se

seleccionaron las formulaciones destacadas en negrita en las Tablas 1.1 y 1.2.

El criterio utilizado para dicha selección se basó principalmente en la menor

2)

Discusión

149

polidispersión de estas formulaciones y en el hecho de que éstas están

elaboradas con una adecuada cantidad de heparina.

En las Tablas 2.1 y 2.2 se presentan los valores de contenido en

heparina, eficacia de asociación y rendimiento de las formulaciones de

quitosano-HA y quitosano-CMβCD. La eficacia de asociación fue, en todos

los casos, similar (~70%), lo que coincide con otros trabajos en los que se

describen nanosistemas de quitosano y heparina 4;99. Esta elevada asociación

se atribuye al alto potencial para interaccionar iónicamente que presentan dos

macromoléculas con carga complementaria, como quitosano y heparina. En

cuanto al contenido de heparina encapsulada, se puede apreciar que este valor

fue siempre mayor para los sistemas que contienen LMWH y, especialmente

alto para las nanopartículas de quitosano-HA (~61%). Ello se puede atribuir a

un mejor ensamblaje de la LMWH en la matriz nanopartícular, lo que puede

inducir un mayor desplazamiento de los demás polianiones (HA, CMβCD,

TPP) que interaccionan con el quitosano y que explicaría también el menor

rendimiento obtenido para los sistemas con LMWH.

Tabla 2. Contenido en heparina, eficacia de encapsulación y de las nanopartículas de a) CS-HA y b) CS-CMβCD (media ± d.e.; n=3).

Cantidad CS-HA-TPP-heparina (mg)

Contenido en heparina (%)

Eficacia de encapsulación (%)

Rendimiento (%)

4-0.6-0.21-1.4a 33.6 ±1.2 72.3 ± 2.7 49.0 ± 1.2

4-0.6-0.21-1.4b 60.6 ± 0.3 69.7 ± 7.6 24.9 ± 4.3 CS= quitosano; a= UFH; b= LMWH.

Cantidad CS-CMβCD-TPP-

heparina (mg)

Contenido en heparina (%)

Eficacia de encapsulación (%)

Rendimiento (%)

6-0.85-0.34-1.6a 38.7 ± 2.5 77.0 ± 2.1 39.4 ± 2.5

6-0.85-0.34-1.6b 44.0 ± 1.1 70.6 ± 2.5 29.3 ± 0.6

99 Chen MC, Wong HS, Lin KJ, Chen HL, Wey SP, Sonaje K, Lin YH, Chu CY, Sung HW. (2009). Biomaterials. 30(34):6629-37.

1)

2)

Discusión

150

Las imágenes de microscopía electrónica (Figura 2) indican que las

formulaciones son esféricas, independientemente del tipo de contraión (HA o

CMβCD) o de heparina utilizados. Es interesante hacer notar que los sistemas

desarrollados con LMWH se ven aparentemente mejor ensamblados que los

que contienen UFH, lo que concuerda con el argumento que acabamos de

exponer.

(1)

(2)

Figura 2. Imágenes de microscopía electrónica de transmisión de las nanopartículas de (1) CS(quitosano)-HA y (2) CS-CMβCD conteniendo a) UFH y b) LMWH.

Estudios de estabilidad de las nanopartículas

Evaluar la estabilidad coloidal de los nanosistemas es un punto clave

para la planificación de estudios posteriores. Para ello se realizaron estudios

de estabilidad con las formulaciones seleccionadas en condiciones similares a

a b

ba

Discusión

151

las utilizadas en cultivos celulares, esto es, a 37°C y utilizando como medios

HBSS a pH 6.4 y 7.4 y PBS a pH 7.4.

La estabilidad de los nanosistemas se valoró a través del seguimiento

de su tamaño en las diferentes condiciones. La composición de los

nanosistemas y de los medios utilizados fueron factores determinantes en su

estabilidad. En el caso de las nanopartículas de quitosano-HA, su tamaño no

experimentó cambios significativos en 24 horas cuando se incubó en PBS a

pH 7.4 (Figura 3), mientras que en HBSS a pH 7.4 sí experimentó un

incremento significativo. A pH 6.4, se agregaron de inmediato, hecho que se

relaciona con el cambio en el potencial zeta de las formulaciones, que

presentó valores cercanos a la neutralidad. A pH 7.4, el potencial zeta de los

sistemas se mantuvo en torno a -10 mV. Esta inversión en la carga superficial

es consecuencia del bajo grado de ionización del quitosano (pKa ~ 6.2) en

medios a pH 7.4. La diferencia entre los perfiles de estabilidad para HBSS

pH 7.4 y PBS pH 7.4 tiene que ver con la distinta composición de estos

tampones (HBSS posee iones CO32- y una concentración de iones PO4

2- 8

veces superior).

En el caso de los sistemas de quitosano-CMβCD, la estabilidad se

mantuvo hasta 24 h en HBSS pH 6.4 (Figura 4), mientras que en los otros

medios las nanopartículas se agregaron inmediatamente. En HBSS pH 6.4 el

potencial zeta de las formulaciones era cercano a +14 mV, mientras que a pH

7.4, los valores fueron neutros (~0 mV), lo que confirma la desestabilización.

Discusión

152

Figura 3: Evolución del tamaño de las nanopartículas de quitosano-HA conteniendo heparina, en medios a pH 7.4: UFH (▲) y LMWH (×) en PBS; UFH (■) y LMWH (□) en HBSS (media ± d.e., n=3).

Figura 4: Evolución del tamaño de las nanopartículas de quitosano-CMβCD conteniendo UFH (■) y LMWH (×) en medio HBSS pH 6.4 (media ± d.e., n=3).

Finalmente, todas las formulaciones seleccionadas mantuvieron su

tamaño sin alteraciones apreciables durante 3 meses en suspensión acuosa a 4

°C (resultados no mostrados).

Estudios de liberación de heparina a partir de las nanopartículas

Se evaluó la liberación de UFH y LMWH a partir de las

formulaciones seleccionadas, en los medios en que éstas alcanzaron la

Discusión

153

máxima estabilidad, esto es, en PBS pH 7.4 para los sistemas de quitosano-

HA y en HBSS pH 6.4 para los sistemas de quitosano-CMβCD.

Considerando la alta densidad de carga negativa que posee la heparina y la

carga positiva del quitosano, se espera que la liberación del fármaco a partir

de la matriz de los nanosistemas sea lenta, como consecuencia de una fuerte

interacción electrostática entre las macromoléculas con carga opuesta98.

Como se puede apreciar en las Figuras 6a y 6b, si bien la liberación

de las heparinas fue lenta en todos los casos, su perfil varió

significativamente dependiendo de su peso molecular y de la composición de

cada nanosistema. En el caso de los sistemas de quitosano-HA, la UFH se

liberó muy lentamente alcanzándose una liberación final de un 10% tras 12 h

de incubación. La LMWH se liberó de una manera más rápida y continua

alcanzándose una liberación final de aproximadamente un 80% (Figura 6a).

a)

Discusión

154

Figura 6: Perfiles de liberación de UFH (□) y LMWH (■) a partir de las nanopartículas de a) CS(quitosano)-HA en PBS pH 7.4; y b) CS-CMβCD en HBSS pH 6.4 (media ± d.e., n=3).

La liberación más rápida de la LMWH se atribuye a su menor peso

molecular, que facilitaría la mejor difusión de la heparina. También debe

tomarse en consideración la gran diferencia en los valores de contenido en

heparina de los sistemas desarrollados con UFH y LMWH (~34% y ~61%,

respectivamente), que apoyaría los resultados expuestos.

Por otro lado, como se puede apreciar en la Figura 6b, la cinética de

liberación de las heparinas a partir de las nanopartículas de quitosano-

CMβCD se ajustó a una pauta diferente. Los nanosistemas conteniendo

LMWH, liberaron el fármaco de manera muy lenta durante la primera hora (~

10%), tras lo cual apenas hay cambios hasta las 12 h de duración del estudio.

La UFH se liberó de una manera más rápida y continua, con una liberación

final de aproximadamente un 50%. La menor liberación observada para la

LMWH puede relacionarse con un mejor ensamblamiento de ésta molécula

en la matriz de los sistemas, como ya se sugirió previamente en relación al

tamaño de los nanosistemas (221 vs 375 nm).

b)

Discusión

155

Estudio de la interacción entre las nanopartículas conteniendo heparina

y los mastocitos

Considerando que la heparina previene la desgranulación de los

mastocitos debido a la interacción que establece con el receptor intracelular

de trifosfato de inositol (IP3)100;101;102, es importante elucidar si su inclusión

en los nanosistemas de quitosano-HA y quitosano-CMβCD facilitará el

acceso de este fármaco al interior de los mastocitos. Para hacer un

seguimiento de las nanopartículas fluorescentes en contacto con mastocitos

de rata, se utilizó la microscopía confocal.

El solapamiento de la señal verde transmitida por las nanopartículas

conteniendo UFH, con la señal roja transmitida por los mastocitos, resultó en

un color naranja (Figura 7). Esto nos confirma que las nanopartículas,

independientemente de su composición, son capaces de interaccionar con los

mastocitos tras un periodo de incubación de 2 h.

a b c d

Figura 7. Imágenes de microscopía confocal de mastocitos fluorescentes tras un contacto de 2 horas con nanopartículas fluorescentes de (1) CS-HA y (2) CS-CMβCD conteniendo UFH: (a)

100 Ahmed T, Ungo J, Zhou M, Campo C. (2000). J. Appl. Physiol. 88:1721–1729. 101 Wong WS, Koh DS. (2000). Biochem. Pharmacol. 59:1323–1335. 102 Niven AS, Argyros G. (2003). Chest 123:1254–1265.

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señal transmitida por los mastocitos (rojo); (b) señal transmitida por las nanopartículas (verde); (c) solapamiento de ambas señales (naranja) y (d) señal óptica.

Para confirmar que esta interacción transcurre a nivel intracelular, y

no sólo a nivel superficial, se obtuvieron secuencialmente planos del eje “z”

de los mastocitos tras su incubación con las nanoparticulas (Figura 8). En

estas imágenes se puede apreciar que, efectivamente, las nanopartículas son

capaces de acceder al interior de los mastocitos distribuyéndose aquí de una

manera relativamente homogénea. Esta información coincide con lo

observado en otros trabajos en los que se ha demostrado, por esta y otras

técnicas, que las nanopartículas que poseen una cubierta de quitosano son

capaces de acceder al interior del espacio celular103;104;105;106, en diferentes

cultivos celulares. Sin embargo, ésta es la primera vez que, utilizando

microscopia confocal, se evalúa y demuestra que las nanopartículas son

internalizadas en mastocitos. Finalmente, los nanosistemas conteniendo

LMWH demostraron un comportamiento similar a los sistemas cargados con

UFH (imágenes no mostradas).

103 Ma Z, Lim LY. (2003). Pharm. Res. 20(11):1812-9. 104 Csaba N, Köping-Höggård M, Fernandez-Megia E, Novoa-Carballal R, Riguera R, Alonso MJ. (2009). J. Biomed. Nanotechnol. 5(2):162-71. 105 Lee DW, Yun KS, Ban HS, Choe W, Lee SK, Lee KY. (2009). J. Control Rel.139(2):146-52. 106 Raviña M, Cubillo E, Olmeda D, Novoa-Carballal R, Fernandez-Megia E, Riguera R, Sánchez A, Cano A, Alonso MJ. (2010). Pharm. Res. 27(12):2544-2555.

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Figura 8. Imágenes de microscopía confocal de mastocitos de rata fluorescentes tras 2 horas de incubación con nanopartículas fluorescentes de (1) CS-HA y (2) CS-CMβCD conteniendo UFH. Planos de los mastocitos tomados cada 1.5 μm del eje “z”.. Señal de exitación para mastocitos (rojo); señal de exitación para nanopartículas (verde).

Estudio de viabilidad celular

La viabilidad de los mastocitos tras su extracción a partir de las

cavidades peritoneales y pleurales de rata fue, en todos los casos, superior a

un 90%. Ésto se comprobó mediante microscopia óptica empleando el

método del azul tripano107. El método utilizado para la extracción de los

107 Oyarzun-Ampuero FA, Brea J, Loza MI, Torres D, Alonso MJ. (2009). Int. J. Pharm. 381:122–129.

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mastocitos fue el descrito por Lago y col.108, y los valores de viabilidad

obtenidos fueron similares a los obtenidos previamente109. Tras un tiempo de

2 horas de contacto con los nanosistemas, la viabilidad fue siempre superior

al 90%, lo que indica la inocuidad de los nanosistemas en el modelo celular

ensayado. La cantidad de nanopartículas utilizada en estos estudios,

correspondió a la más alta que sería administrada a los mastocitos en los

estudios de liberación de histamina que se presentan a continuación.

Estudio de inhibición de la liberación de histamina en mastocitos

Como método de evaluación de la eficacia antiasmática de la

heparina encapsulada, se estudió su capacidad para prevenir la liberación de

histamina por parte de los mastocitos. De acuerdo con la información que

disponemos, ésta es la primera vez que este tipo de experimentos se lleva a

cabo con nanosistemas.

Estudio preliminar: En primer lugar, se evaluó el efecto de diferentes dosis

de UFH y LMWH en solución para prevenir la liberación de histamina

inducida por el compuesto estándar de origen sintético 48/80. El compuesto

48/80 es conocido por su capacidad de desgranulación de los mastocitos,

promoviendo así la liberación al medio extracelular de la histamina

localizada en el interior de dichos gránulos110. En la Figura 9 se puede

apreciar que tanto la UFH como la LMWH poseen, en el rango de

concentración ensayado, un efecto dosis dependiente similar, para prevenir la

liberación de histamina (IC50 μg/mL = 6.8±1.2 para UFH y 12.3±3.1 para

LMWH).

108 Lago J, Alfonso A, Vieytes MR, Botana LM. (2001). Cell Signal. 13:515–524. 109 Buceta M, Dominguez E, Castro M, Brea J, Alvarez D, Barcala J, Valdes L, Alvarez-Calderon P, Dominguez F, Vidal B, Diaz JL, Miralpeix M, Beleta J, Cadavid MI, Loza MI. (2008). Biochem. Pharmacol. 76, 912–921. 110 Ortner MJ, Chignell CF. (1981). Immunopharmacology 3:187–191.

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0

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Compuesto 48/80

Solución UFH

Solución LMWH

Figura 9. Efecto de la concentración de UFH y LMWH en solución sobre la liberación de histamina en mastocitos. La liberación de histamina se inició tras la incubación de las células con una solución 100 μM del compuesto 48/80. Las células se preincubaron con diferentes concentraciones de UFH y LMWH antes de la adición del compuesto (n=3, p< 0.05).

Se confirmó que en las condiciones del ensayo, la heparina es capaz

de prevenir la liberación de histamina y, por otro lado, evidencia el rango

efectivo de concentraciones de UFH y LMWH. Ambos tipos de heparina

inhibieron la liberación de histamina en una magnitud similar, sin apreciarse

diferencias significativas entre ellas. Ésto contrasta con la información

presentada en diferentes publicaciones en las que se evidencian diferencias

significativas entre los efectos de las heparinas inhaladas de alto y bajo peso

molecular para prevenir la liberación de la histamina111;112;113. De hecho, se

indica que la potencia de las distintas heparinas ensayadas es inversamente

proporcional a su tamaño molecular. La diferencia entre estos resultados

puede deberse a la comparación del contacto directo de las heparinas con los

111 Ahmed T, Campo C, Abraham MK, Molinari JF, Abraham WM, Ashkin D, Syriste T, Andersson LO, Svahn CM. (1997). Am. J. Respir. Crit. Care Med. 155(6):1848-55. 112 Martinez-Salas J, Mendelssohn R, Abraham WM, Hsiao B, Ahmed T. (1998). J. Appl. Physiol. 84(1):222-8. 113 Campo C, Molinari JF, Ungo J, Ahmed T. (1999). J. Appl. Physiol. 86(2):549-57.

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mastocitos ex vivo, con la administración in vivo de las heparinas por

nebulización. En el último caso, las características propias de la fisiología

pulmonar (aclaramiento mucociliar, actividad enzimática, etc.) pueden

influenciar de manera significativa el efecto de una u otra heparina.

Efecto de las nanopartículas conteniendo heparina sobre la liberación de

histamina en mastocitos: En el caso de los sistemas de quitosano-HA

conteniendo UFH (Figura 10a), se evidencia un efecto dosis-dependiente

sobre la inhibición de la liberación de histamina, sin apreciarse diferencias

significativas entre la formulación nanoparticular y el control de heparina en

solución.

En el caso de los sistemas de quitosano-CMβCD conteniendo UFH

(Figura 10b), también se observa un efecto dosis-dependiente para prevenir la

liberación de histamina. Sin embargo, se evidencia una mejora significativa

en este parámetro cuando se ensaya la dosis más alta de UFH (200 μg/mL),

que es capaz de inhibir casi completamente el efecto estimulante de

degranulación del compuesto 48/80 (~90% de inhibición), mientras que la

misma dosis de UFH en solución inhibe sólo un 55%.

Resultados muy similares a los presentados en el presente apartado se

obtuvieron cuando la LMWH estaba contenida en sistemas de quitosano-HA

y quitosano-CMβCD (Capitulo 2, Figura 6b y Capitulo 3, Figura 5b).

Discusión

161

0

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Compuesto 48/80

Nanopartículas blancas de CS-HA

Solución UFH

Nanopartículas de CS-HA con UFH

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Solución UFH

Nanopartículas de CS-CMβCD con UFH

Figura 10. Efecto de la heparina encapsulada en las nanopartículas de (a) CS(quitosano)-HA y (b) CS-CMβCD sobre la liberación de histamina en mastocitos. La liberación de histamina se inició tras la incubación de las células con una solución 100 μM del compuesto 48/80. Las células se preincubaron con diferentes concentraciones de UFH en solución (barras grises) y UFH encapsulada en las nanopartículas (barras crema). Como control, se incubaron también las nanopartículas blancas (n=3, p< 0.05).

Como control negativo de los experimentos, y en todos los casos, se

evaluó el efecto las formulaciones nanoparticulares blancas, sobre la

liberación de histamina, apreciándose que ningún sistema per se tiene un

efecto significativo sobre este parámetro.

*

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Los resultados presentados evidencian la importancia que tiene la

composición de las nanopartículas sobre el efecto de la heparina sobre

mastocitos. En el caso de las nanopartículas de quitosano-CMβCD,

independientemente del peso molecular de la heparina, se observó siempre

una mejora significativa sobre el efecto obtenido con la heparina en solución.

Nuestra hipótesis es que dicha mejora se relaciona con un efecto promotor de

la CMβCD en el acceso intracelular de la heparina en mastocitos, que se hace

notar a la concentración más alta de heparina, con la que hay también una

mayor concentración de ciclodextrinas. Una situación diferente se presenta

con los nanosistemas de quitosano-HA, que no llegaron a demostrar, al

menos a las dosis ensayadas, un efecto superior al de la heparina en solución,

lo que indica una menor capacidad internalizante. De cualquier modo, resulta

complicado comparar las dos formulaciones de nanopartículas, ya que

además de las diferencias atribuidas a las propiedades de los contraiones, HA

y CMβCD, existen otras diferencias que pueden tener un efecto significativo

sobre la eficacia de la heparina, como por ejemplo la relación final entre

polímeros y heparina, valor difícil de estimar, al ser diferentes los

rendimientos obtenidos para cada formulación.

Otras diferencias significativas entre las formulaciones en parámetros

como el tamaño, los perfiles de estabilidad y liberación de la heparina,

pueden también condicionar el efecto biológico de la heparina.

Finalmente, es importante considerar que las condiciones ex vivo

utilizadas en los experimentos no reflejan las barreras fisiológicas de las vías

aéreas como son el aclaramiento mucociliar y la actividad enzimática. Dichas

barreras pueden ser superadas eficazmente por los nanosistemas propuestos,

debido a la demostrada propiedad mucoadhesiva del quitosano114;115 y del

114 Aspden TJ, Mason JD, Jones NS, Lowe J, Skaugrud O, Illum L. (1997). J. Pharm. Sci. 86: 509–513. 115 Lim ST, Martin GP, Berry DJ, Brown MB. (2000). J. Control. Rel. 66, 281–292.

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HA9;116 y a la capacidad de las nanopartículas para proteger el fármaco

encapsulado de la actividad enzimática. Un buen ejemplo en el que se

demuestra la eficacia de las nanopartículas recubiertas con quitosano para

promover el efecto localizado de un fármaco macromolecular administrado

por vía pulmonar, ha sido previamente publicado por Bivas-Benita y col.

(2004)117. El enfoque del citado trabajo fue el desarrollo una vacuna

conteniendo un plásmido de DNA para prevenir la tuberculosis.

De manera adicional, las formulaciones nanoparticulares propuestas

pueden mejorar el efecto de una formulación convencional de heparina

debido a su potencial para liberar lentamente el fármaco, lo que prolongaría

su efecto antiasmático. Desafortunadamente, las condiciones experimentales

ex vivo utilizadas en el presente trabajo no permiten realizar experimentos de

mayor duración, lo que no permite verificar esta teoría.

116 Pritchard K, Lansley AB, Martin GP, Helliwell M, Marriot C, Benedetti LM. (1996). Int. J. Pharm. 129:137–145. 117 Bivas-Benita M, van Meijgaarden KE, Franken KL, Junginger HE, Borchard G, Ottenhoff TH, Geluk A. (2004). Vaccine 22(13-14), 1609-15.

PARTE II: DESARROLLO DE UN NUEVO SISTEMA CONSTITUÍDO

POR NANOCÁPSULAS DE ÁCIDO HIALURÓNICO CONTENIENDO

DOCETAXEL Y EVALUACIÓN DE EFICACIA ANTITUMORAL

SOBRE CULTIVOS CELULARES DE CÁNCER DE PULMÓN.

Capitulo 4

Hyaluronan nanocapsules: a new safe and effective nanocarrier for the

intracellular delivery of anticancer drugs

Capitulo 4

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Abstract

Here we report a new drug nanocarrier for the intracellular delivery

of hydrophobic anticancer drugs. The nanocarrier -named as nanocapsules- is

composed of a lipid core surrounded by a shell made of hyaluronic acid

(HA). This polymer was chosen because of its ability to prolong the

circulation time and the interaction with certain cancer tumors. HA

nanocapsules were produced by a modified solvent displacement technique,

which allows the formation of the polymer shell around the oily core, as a

consequence of the interaction between a cationic surfactant and the anionic

polysaccharide. The resulting HA nanocapsules have average sizes from 170

to 250 nm and a zeta potential values ranging from -30 and -60 mV. They

showed a capacity to efficiently encapsulate the hydrophobic drug docetaxel

(DCX) and retain it in the oily core upon dilution in a simulated biological

fluid. As expected, the nanoencapsulated DCX exhibited an enhanced

cytotoxicity (3-fold increase as compared to a control solution) upon in vitro

incubation with the cancer cell line NCI-H460. This result was attributed to

the internalization of the nanocapsules and the intracellular delivery of DCX.

Moreover, the nanocapsules suspension could be freeze-dried and remained

stable during storage. In summary, these novel nanostructures hold promise

as intracellular drug delivery systems.

Keywords: Nanocapsules, hyaluronic acid, cancer, hydrophobic drugs,

intracellular delivery.

Capitulo 4

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Introduction

Currently, it is known that the use of nanoscale drug delivery

vehicles represents a very promising strategy for improving the

biodistribution and intracellular delivery of anticancer drugs. Taxanes

(paclitaxel, docetaxel) are good examples of potent chemotherapeutic agents,

which could greatly benefit from these delivery carriers. Indeed, despite their

efficacy, these drugs display significant draw-backs related to their

indiscriminate biodistribution and the necessity to use toxic solubilizers for

their intravenous administration (Ten Tije et al., 2003). Besides the marketed

formulation of albumin nanoparticles (Abraxane), a number of nanocarriers

have been disclosed in the literature for the delivery of these specific

compounds. Particularly attractive for this purpose are the nanocapsules,

which can easily accommodate hydrophobic (Heurtault et al., 2002a, b; Bae

et al., 2007; Lozano et al., 2008; 2011b). For example, the group of Benoit

and co-workers has reported the potential of PEG-coated lipid nanocapsules

loaded with paclitaxel in different cancer animal models. Overall, the authors

observed that PEGylated nanocapsules have long circulating properties as

well as the ability to improve the intracellular accumulation of drugs in the

tumor cells (Garcion et al., 2006; Lacoeuille et al., 2007). An alternative

nanocapsule-type carrier was recently reported by our group for the delivery

of docetaxel (DCX) (Lozano et al., 2008; 2011b). These nanocapsules are

made of chitosan and polyarginine and display a number of properties,

among which it is important to highlight: I) their capacity to be internalized

by different cell lines, such as the breast cancer (MCF-7) and lung cancer (A-

549) cell lines, II) the improved efficacy of DCX-loaded nanocapsules in

MCF-7, A-549 and NCI-H460 (non small cell lung cancer) cell lines

compared with DCX alone; and III) the possibility to functionalize them with

ligands such as anti-TMEFF-2 in order to target them to TMEFF-2, a

Hyaluronan nanocapsules: a new safe and effective nanocarrier for the intracellular delivery of anticancer drugs

172

transmembrane protein, that is overexpressed in non microcytic tumors

(Torrecilla et al., 2011).

Following this experience, we found it important to modify the

polymer corona of the nanocapsules by using hydrophilic negatively charged

polymers such as hyaluronic acid (HA). This polysaccharide attracted our

attention because of its reported ability to prolong the plasma circulation time

of liposomes and nanoparticles (Peer and Margalit, 2004a, b; Peer et al.,

2007; Choi et al., 2009; Ossipov, 2010). Additionally, HA offers the

possibility of targeting the drug-loaded nanocarrier to the cancer cells that

overexpress the endogenous receptor for this polymer (called CD-44

receptors). As a consequence of these properties, HA-based nanocarriers have

resulted in an improved tumor growth inhibition and a decreased systemic

toxicity, when compared to the free drug (Akima et al., 1996; Luo and

Prestwich., 1999; Elias and Szocka., 2001; Eliaz et al., 2004; Peer and

Margalit., 2004 a, b; Rosato et al., 2006; Auzenne et al. 2007; Hyung et al.,

2008).

Taking this background information into account our aim in this

work has been to develop a new and alternative HA-based anticancer drug

delivery nanocarrier exhibiting unique pharmaceutical properties such as: I)

acceptable from the safety point of view, II) able to load important amounts

of lipophilic anticancer drugs, III) ability to target and enter cancer cells

providing intracellular drug delivery and, finally, IV) easy to produce and

scale-up and stable under storage. The results presented in this report clearly

show that HA nanocapsules fulfill such interesting profile.

Capitulo 4

173


Chemicals: Docetaxel (DCX, from Fluka), poloxamer (Pluronic F-

68®), benzalkonium chloride (BKC) and hexadeciltrymethylammonium

bromide (CTAB) were purchased from Sigma-Aldrich (Spain). Miglyol

812®, which is neutral oil formed by esters of caprylic and capryc fatty acids

and glycerol, was donated by Sasol Germany GmbH (Germany). The

surfactant Epikuron 145V, which is a phosphatidylcholine-enriched fraction

of soybean Lecithin, was donated by Cargill (Spain). Hyaluronic acid of 29

kDa was purchased from Imquiaroma (Barcelona, Spain) and that of 160 kDa

was kindly donated by Bioiberica (Barcelona, Spain).

Preparation of HA nanocapsules:

HA nanocapsules were prepared following two different procedures

that have been previously described by our group (Prego et al., 2005; Lozano

et al., 2008). The first one, called “two-stage procedure” consist in adding

125 μL of Miglyol 812 to an organic phase comprising 30 mg of lecithin, and

4 mg of BKC or 1.8 mg of CTAB, dissolved in 0.5 ml of ethanol and 9.5 ml

of acetone. This organic phase was added to an aqueous phase (20 mL). The

formation of the cationic nanoemulsions was instantaneous, which was

evident due to the milky appearance of the mixture (interestingly, the cationic

nanoemulsion was also obtained after the addition of the organic phase to a

homogenizer/sonicator, data not shown). The above solution was

rotaevaporated until a volume of 10 mL, and then incubated with an aqueous

solution of HA (0.1-50 mg) in a volume ratio 4:1.5 (cationic

nanoemulsion:HA solution); when the anionic HA interacts with the cationic

nanoemulsion, it forms a polymer corona at the oil/aqueous interface thus

originating HA nanocapsules. The second one, called “one-stage procedure”

involves adding 125 μL of Miglyol 812 to an organic phase comprising 30


174

mg of lecithin, and 4 mg of BKC or 1.8 mg of CTAB, dissolved in 0.5 ml of

ethanol and 9.5 ml of acetone. This organic phase was added to an aqueous

phase (20 mL) that contains the polymer HA (0.1-50 mg). The above solution

was rotaevaporated until a volume of 10 mL, here the formation of HA

nanocapsules occurs concomitantly to the evaporation of the solvent due to

adsorption of the polymer onto the nanocarrier-surface. In both cases, the

incorporation of DCX, required the previous dissolution of this molecule in

ethanol to obtain a final concentration of 1 mg/mL. Next, an aliquot of the

stock solution was added to the organic phase and the same procedure was

followed. The final DCX concentration obtained in HA nanocapsules carriers

was 7.27 μM.

Physicochemical Characterization of HA nanocapsules





The morphological examination of the systems was performed by

transmission electron microscopy (TEM) (CM12 Phillips, Netherlands). The

samples were stained with 2% (w/v) phosphotungstic acid and immobilized

on copper grids with Formvar® for viewing by TEM.

Encapsulation Efficiency of DCX-Loaded HA nanocapsules

The encapsulation efficiency of DCX in the nanocarriers was

determined indirectly by the difference between the total amount of DCX and

the free drug recovered in the continuous phase. The total amount of drug

was estimated by dissolving an aliquot of non isolated DCX-loaded HA

nanocapsules with acetonitrile. This sample was centrifuged during 20 min at

Capitulo 4

175

4000 × g and the supernatant was measured with a high performance liquid

chromatography (HPLC) system. The non encapsulated drug was determined

by the same method following separation of the nanocapsules from the

aqueous medium by ultracentrifugation (27500 g, 60 min). DCX was assayed

by a slightly modified version of the method proposed by Lee et al (1999).

The HPLC system consisted of an Agilent 1100 series instrument equipped

with a UV detector set at 227 nm and a reverse phase Zorbax Eclipse XDB-

C8 column (4.6 × 150 mm i.d., pore size 5 μm Agilent, U.S.A.). The mobile

phase consisted of a mixture of acetonitrile and 0.1% v/v orthophosphoric

acid (55:45, v/v) and the flow rate was 1 mL/min. The standard calibration

curves of DCX were linear (r2 > 0.999) in the range of concentrations

between 0.3-2 μg/mL.

In vitro Release Studies

The release studies of DCX from HA carriers were performed by

incubating a sample of the formulation with milli-Q water at an appropriate

concentration to ensure sink conditions. The vials were placed in an incubator

at 37 °C with horizontal shaking. A total of 3 mL of the suspension were

collected and ultracentrifuged (27500 g, 60 min) by using Herolab high speed

centrifuge labware tubes (Herolab GmbH, Germany) at different time

intervals. The DCX released was calculated indirectly by determining how

much of it was left in the system by processing the isolated HA nanocapsules

with acetonitrile before HPLC analysis.


176

Cellular assays

Cell viability assay and IC50 estimation: Human non-small cell lung

cancer cell line NCI-H460 was cultured in RPMI 1640 medium (ATCC),

supplemented with 10% (v/v) fetal bovine serum (FBS) at 37° C in a

humidified atmosphere containing 5% carbon dioxide. Tretazolium salt 3-

(4,5-dimewthylthiazol-2-yl)2,5 diphenyltetrazolium bromide (MTT, Acros

Organics) was used for mytocondrial activity evaluation. Briefly, cells were

plated onto 96-well plates, with a seeding density of 15x103 cells/well in 100

μL culture medium. After 24 h, the medium was renewed but containing the

following three treatments: DCX, DCX- loaded HA nanocapsules and blank

HA nanocapsules. Finally, after 48 h cell survival was measured by the MTT

assay (Mossmann., 1983). In brief, medium was removed and the cells were

washed twice with 100 μL Hank`s Balanced Salt Serum (HBSS). Then 20 μL

of a MTT solution (5 mg/mL in PBS) and 100 μL HBSS were added to the

wells and maintained at 37°C in an atmosphere with 5% CO2 for 4h.

Afterwards, buffers were replaced by 100 μL DMSO per well and maintained

at 37° C in an atmosphere with 5% CO2 overnight. Absorbance (λ= 515 nm)

was measured in a spectrophotometer (Tecan, Ultra evolution) removing

background absorbance (λ= 630 nm). Moreover, short incubation times of 2 h

were assayed in order to determine the efficacy of HA nanocapsules to

quickly interact with the cells and deliver the drug intracellularly. Thus, after

2 h of incubation with the three treatments, medium was replaced by fresh

one and cells were grown for 48 h. Finally, cell viability was measured as

described.

The percentage of cell viability was calculated by the absorbance

measurements of control growth in the presence of the formulations at

various concentration levels. IC50 values were obtained by fitting the data

with non linear regression, with Prism 2.1 software (GraphPad, San Diego,

CA).

Capitulo 4

177

Stability study at storage conditions

The suspension stability of HA nanocapsules prepared with the

surfactants BKC and CTAB were evaluated according to terms of time and

temperature of storage. Therefore, aliquots of the nanocapsules suspension

without dilution were placed in sealed tubes at 4 and 37°C for storage. Size

and polydispersity index of the nanocarriers were measured for a period of

three months, meanwhile zeta potential values were controlled at the end of

the study. Each sample corresponds to a different HA nanocapsules batch.

Freeze-dried studies of HA nanocapsules

Concentrations of HA nanocapsules (0.25, 0.5 and 1% w/v) and of

the cryoprotectant trealose (5 and 10%) were considered the variables for the

lyophilization study. Therefore, 2 mL dilutions of HA NCs were transferred

in 5 mL volume glass vials and were frozen at -20°C. The lyophilization

procedure consisted in an initial drying step for 60 h at -35°C, followed by a

secondary drying for 24 h in a high vacuum atmosphere. Finally, temperature

was slowly increased up to 20°C until the end of the process (Labconco

Corp., USA). HA nanocapsules were recovered by adding 2 mL of ultrapure

water ot the freeze-dried powders followed by manual resuspension and were

characterized as explained above.


178

Statistical analysis

Cell culture results were evaluated in order to determine the

statistical significance between the different formulations studied. The

statistical evaluation of the cell viability results was performed by an

ANOVA test followed by the post hoc Tukey test comparison analysis

(SigmaStat Program, Jandel Scientific, version 3.5).


The main aim of this work was the development of a new anticancer

drug nanocarrier consisting of a lipid core surrounded by a shell made of HA.

The conceptual bases of the system were: simplicity (minimum amount of

components and easy to produce), acceptability (from the regulatory stand

point) and efficacy (improvement of citoxicity in cancer cells). For this, DCX

was chosen as a model compound and its efficacy was evaluated in lung

cancer cell line NCI-H460. As indicated in the introduction, the biopolymer

HA was chosen because of its interesting biopharmaceutical properties (Eliaz

and Szoka, 2001; Surace et al., 2009; Ossipov et al., 2010).

Development and characterization of HA nanocapsules

HA nanocapsules were prepared by a number of simple techniques,

which involved the formation of a cationic nanoemulsion and the attachment

of the outer HA corona. One of the techniques was the solvent displacement

technique, which allowed the emulsification process to occur simultaneously

to the attachment of the polymer corona. Other techniques involved two

steps, i.e. an emulsification process either by the solvent displacement

technique, homogenization or sonication, followed by the coating with the

polymer. In all cases, the formation of the polymer capsule is driven by the

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179

ionic interaction of the positively charged surfactant with the negatively

charged HA. Following an initial screening of different experimental

approaches, we chose the two-step solvent diffusion technique previously

used for the formation of chitosan (Lozano et al., 2008) and polyarginine

nanocapsules (Lozano et al., 2011b), and investigated the influence of

different formulation parameters on the physicochemical properties of the

resulting systems. The formulation parameters were the type and

concentration of cationic surfactant, and the concentration of HA. The

cationic surfactants selected on the basis of their acceptable toxicological

profile (Rowe et al., 2009) were BKC and hexadecyltrimethylammonium

bromide (CTAB). On the other hand, the quantity of the surfactants used was

the minimum that allowed us the formation of stable systems. In the case of

CTAB this amount was 1.8 mg, and in the case of BKC 4.0 mg.

Table 1a and b show the characteristics of nanocapsules prepared by

the two-stage procedure using the surfactants BKC or CTAB, respectively.

As can been noted, the use of adequate concentrations of HA with the

cationic nanoemulsions results in the formation of homogenous populations

of nanocapsules of around 250 nm. The results also show a shielding of the

original positive zeta potential of the nanoemulsion, which leads to an

inversion to negative values, as the concentration of HA increases. This

dependency of the zeta potential with the amount of HA evidences the

surface localization of HA molecules and indicates the necessity to use a

minimum amount of HA in order to obtain a stable nanocapsules. The

formation of the HA corona results in a minor increase in the size of the

nanocapsules with respect to the nanoemulsions, a result that supports the

idea that HA is tightly attached to the oily droplets.


180

Tables 1a and b also indicate that the viability of the system is

compromised under certain conditions. More specifically, we observed a

precipitation of the system due to the neutralization of the positive surface

charge caused by the addition of increasing amounts of HA. After the

inflection point, the addition of subsequent amounts of HA leads to the

inversion of the zeta potential and the subsequent stabilization of the system.

Finally, there is another precipitation point that is related to the excess of the

HA in the formulation. Here, the precipitation phenomenon could be

attributed to a combination of factors such as the high ionic strength in the

medium (Hiemenz and Rajagopalan, 1997) (due to the high concentration of

sodium ions added together with the hyaluronate), combined with the

increase in the viscosity, which might modify the kinetic of adsorption of the

polymer onto the cationic nanoemulsion (Cowman and Matsuoka, 2005).

Table 1. Physicochemical properties of the nanocapsules prepared following a two-stage procedure, with different quantities of HA (29 kDa) and a fixed amount of the surfactants (a) BKC (4.0 mg) or (b) CTAB (1.8 mg ) (mean± S.D., n≥3). (a)

HA 29 kDa (mg) Size (nm) Polydispersity

Index Zeta potential

(mV) 0.0 223.3 ± 10.3 0.1 +27.1 ± 3.5 0.5 249.8 ± 10.2 0.2 +9.6 ± 2.5 1.0 pp. --- --- 3.1 pp. --- --- 6.2 248.7 ± 10.0 0.2 -42.3 ± 2.1 12.5 235.3 ± 13.3 0.1 -45.6 ± 4.7 25.0 252.2 ± 19.1 0.1 -46.7 ± 2.9 50.0 pp. --- ---

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181

(b)



(mV) 0.0 215.2 ± 7.3 0.1 +35.3 ± 2.1 0.1 231.7 ± 8.3 0.1 +18.3 ± 2.2 0.5 pp. --- --- 1 pp. --- ---

3.1 238.2 ± 2.4 0.1 -35.3 ± 2.4 6.2 238.4 ± 3.3 0.1 -37.6 ± 2.2 12.5 260.3 ± 20.0 0.2 -31.3 ± 3.2 25.0 pp. --- --- 50.0 pp. --- ---

pp.: Precipitation.

Tables 2a and 2b display the results of size and zeta potential of the

HA nanocapsules prepared according to a single stage procedure. Overall, the

conclusion is that the incorporation of HA either during or after the

emulsification process does not affect the formation of the HA corona

(similar zeta potential values and precipitation conditions). However, the size

of the nanocapsules elaborated by a single stage procedure was smaller than

that of nanocapsules obtained by a two-stage procedure (~170nm and

~250nm, respectively). This difference in size has also been observed for

chitosan nanocapsules prepared following a single or two-stage procedure

(Prego et al., 2005; Lozano et al., 2008) and could be attributed to the

stabilizing role of the polymer during the emulsification process.


182

Table 2. Physicochemical properties of the nanocapsules prepared following a single stage procedure with different quantities of HA (29 kDa) and a fixed amount of the surfactants (a) BKC (4.0 mg) or (b) CTAB (1.8 mg ) (mean± S.D., n≥3). (a)

HA 29 kDa (mg) Size (nm) Polydispersity Index

Zeta potential (mV)

0.0 223.3 ± 10.3 0.1 +27.1 ± 3.5

0.5 251.2 ± 5.2 0.1 +27.2 ± 1.4 1.0 pp. pp. pp. 3.1 pp. pp. pp. 6.2 pp. pp. pp. 12.5 185.2 ± 4.6 0.1 -44.2 ± 2.3 25.0 186.4 ± 6.6 0.1 -47.4 ± 2.3 50.0 pp. pp. pp.

(b)

HA 29 kDa (mg) Size (nm) Polydispersity Index

Zeta potential (mV)

0.0 215.7 ± 7.2 0,1 +35 ± 2.5 0.1 228.6 ± 17.3 0.1 +33.7 ± 3.0 0.5 pp. pp. pp. 1.0 pp. pp. pp. 3.1 168.4 ± 3.4 0.1 -35.9 ± 2.7 6.2 170.3 ± 6 0.1 -35.6 ± 1.0 12.5 273.4 ± 12.7 0.2 -35.7 ± 2.8 25.0 pp. pp. pp. 50.0 pp. pp. pp.

pp.: Precipitation.

The size and appearance of the nanocapsules has also been observed

by transmission electron microscopy (TEM). As an example, figure 1

illustrates the size and structure of those prepared with the BKC surfactant

(figure 1). Overall, the nanocapsules exhibited a round shape and the core-

corona structure typically observed for polymeric nanocapsules (Prego et al.,

2005; Lozano et al., 2008).

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183

Figure 1: Transmission electron microscopy images of HA nanocapsules obtained with 12.5 mg of HA (29 kDa) and containing BKC (4 mg).

In a second part of the development process, we evaluated the

influence of the HA MW (160 kDa vs. 29 kDa used in the original study) in

the characteristics of the nanocapsules prepared by the two-step solvent

diffusion technique. Table 3 shows the characteristics of the formulations

prepared by incubation of the preformed emulsion containing BKC with high

MW HA. Overall, the values corresponding to size, polidispersity, and zeta

potential are larger than those obtained with HA of 29 kDa (table 1a and b).

Similar results were obtained when the surfactant used was CTAB (data not

shown). This behavior correlates very well with the previously observed for

chitosan nanocapsules (Santander-Ortega et al. 2011) and was attributed to

the different thickness of the HA coating around the oily nanodroplets.

Namely, the use of high MW led to an increase in the thickness of the coating

and, thus, to an increase of the size of the nanocapsules. In addition, an

increase of the zeta potential values was also observed as compared to the

low MW HA nanocapsules, a result that could be a consequence of the

greater number of carboxylic groups of AH at the shell surface.


184

Table 3. Physicochemical properties of the nanocapsules prepared following a two-stage procedure with different quantities of HA (160 kDa), with the surfactant BKC (4 mg) (mean± S.D., n≥3).



(mV) 0.0 207.6 ± 5.5 0.1 +27.3 ± 3.1 0.5 235.2 ± 10.3 0.2 +27.3 ± 3.6 1.0 271.4 ± 53.8 0.5 +3.9 ± 1.8 3.1 pp. --- --- 6.2 256.6 ± 11.4 0.5 -52.4 ± 8.8

12.5 294.5 ± 40.6 0.4 -62.3 ± 8.1 25.0 410.7 ± 70.0 0.5 -59.9 ± 6.1 50.0 pp. pp. pp.

pp.:Precipitation.

Encapsulation and release of docetaxel (DCX) from HA nanocapsules

The high efficacy of DCX in the treatment of a wide range of solid

tumors (Crown and O'Leary., 2000), together with its hydrophobic character

makes this molecule an attractive candidate for inclusion in the developed

nanocapsules. Among the tested blank nanocapsules formulations, we

selected the one containing 12.5 mg of 29 KDa HA (see tables 1a and 1b) for

the further studies as it contained the greatest amount of HA without causing

precipitation. As shown in table 3, DCX could be efficiently encapsulated in

the HA nanocapsules prepared with the surfactants BKC or CTAB hardly

affecting the characteristics of the original blank formulations. These data of

encapsulation correlates very well with previously published works where

DCX was also encapsulated in lipid nanocapsules, and is attributed to the

affinity of the drug for the core components (Khalid et al., 2006, Lozano et

al., 2008).

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185

Table 3. Characteristics of the selected blank and DCX-loaded HA nanocapsules prepared with the surfactants BKC or CTAB. (mean ± S.D., n≥3).

Formulation Size (nm) Polydispersity


(mV) Association

efficiency (%) HA NCs (BKC) 235.5 ± 13.2 0.1 -45.0 ± 4.3 ---

DCX- loaded HA NCs (BKC)

250.3 ± 20.5 0.1 -52.3 ± 11.3 65.5 ± 3.1

HA NCs (CTAB) 267.2 ± 23.4 0.1 -31.1 ± 3.4 --- DCX- loaded HA

NCs (CTAB) 276.2 ± 10.3 0.1 -36.7 ± 1.4 65.1 ± 3.8

In an additional experiment, we evaluated the release pattern of the

encapsulated DCX upon incubation of highly diluted nanocapsules prepared

with the surfactants BKC or CTAB in aqueous medium. The results showed

similar drug release profiles for nanocapsules containing BKC or CTAB with

only critical differences in the initial time-release point (figure 2). The release

follows a biphasic profile, characterized by a rapid initial release, followed

by a second stage in which no further changes were observed. The initial

release stage, which is typically observed in oily systems (Prego et al., 2006;

Lozano et al., 2008; 2011b), has been attributed to the dilution of the

nanocapsules in the incubation medium and the subsequent partition of the

drug between the oily core and the external aqueous phase. The absence of

release in the second stage confirms the high affinity of the DCX by the oil

core. While these data provide us with information about mechanistic details,

in their interpretation it is important to point out that the release behavior

observed in vitro is not expected to correlate with the in vivo behavior. In the

in vivo situation, the presence of physiological occurring macromolecules and

ions could significantly influence the release profile as reported Ahmed et al

(2011).


186

Figure 2: In vitro DCX release from DCX- loaded HA nanocapsules using BKC (X) or CTAB ( ) as surfactants (mean± S.D., n=3).

In vitro efficacy of the DCX- loaded HA nanocapsules in human lung

cancer cell line

Cell viability studies were performed in order to assess the efficacy

of the HA nanocapsules in the non-small cell lung cancer NCI-H460 cell line.

Figures 3a and b show the cellular viability profiles of DCX-loaded HA

nanocapsules (formulation containing 1.8 mg of CTAB and 12.5 mg of HA

29 KDa) upon cell exposure for up to 2 and 48 h respectively. The results

indicate that the encapsulation of DCX in HA nanocapsules leads to a

significant increase of its citotoxicity. The toxicity values were multiplied by

a factor of 2 or 3 depending upon the incubation time. The results also show

that blank nanocapsules do not cause any noticeable damage to the cells.

These results are summarized in table 4, which illustrates the citotoxicity

values of DCX-loaded nanocapsules measured by their IC50. Overall, the

potency of DCX was increased up to more than 3 times upon its

encapsulation into HA nanocapsules.

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187

Figure 3: Effect of DCX encapsulation into HA nanocapsules (HA= 29 KDa, 12.5 mg) containing CTAB (1.8 mg) on NCI-H460 cell viability after (a) 2 or (b) 48 h incubation. Blank HA NCs (white bars), free DCX (grey bars) and DCX- loaded HA NCs (black bars) (n=4). # p<0.005: DCX- loaded HA NCs vs. free DCX. (a)

(b)


188

Table 4: IC50 values (drug concentration resulting in a 50% of cell viability) expressed in nM. Mean values of 4 independent experiments; n.d.: none of the concentrations tested resulted in a significant reduction in the viability.

Formulation 2 h exposure 48 h exposure

Blank HA CTAB NCs n.d. n.d. Blank HA BKC NCs n.d. *

DCX 105.0 ± 9.0 36.4 ± 4.0 DCX- loaded HA CTAB NCs 31.5 ± 2.1# 10.8 ± 1.1# DCX- loaded HA BKC NCs 29.1 ± 1.8# 15.3 ± 2.0#

* The maximum reduction in cell viability was 39.0 ± 8.0%. # P<0.01 with respect to IC50 of DCX (One-way ANOVA test, post-hoc Tukey test).

Figures 4a and b show the cytotoxicity profiles of cell exposed to HA

nanocapsules prepared with the surfactant BKC for up to 2 and 48 h,

respectively. The cellular behavior displayed in figure 4a is similar to that

observed for nanocapsules prepared with the surfactant CTAB. Accordingly,

the results in table 4 show that DCX-loaded nanocapsules are 3.6 times more

efficient than the free drug in terms of the IC50 values. This improved

efficacy was also observed after 48 h incubation (figure 4b), leading to a 3-

fold reduced IC50 value (table 4). In this case, a certain reduction of cell

viability was also observed at high concentrations of blank nanocapsules

(figure 4b), a result that was attributed to the higher toxicity of BKC over

CTAB (Rowe, 2009) and the necessity of using a greater amount of BKC vs.

CTAB for the formation of stable nanocapsules.

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189

Figure 4: Effect on NCI-H460 cell viability of HA nanocapsules (AH= 29 KDa, 12.5 mg) prepared with the surfactant BKC (4 mg) after (a) 2 or (b) 48 h of contact with the cells. Blank HA NCs (white bars), free DCX (grey bars) and DCX- loaded HA NCs (black bars) (n=4). # p<0.005: DCX- loaded HA NCs vs. free DCX.

The significant improvement in cytotoxicity observed for HA

nanocapsules loaded with DCX is in agreement with the results observed for

other nanocapsules coated with polyethyleneglycol (Garcion et al., 2006),

chitosan (Lozano et al., 2008) or polyarginine (Lozano et al., 2011b) tested in

a variety of cell lines, including: glyoma cells (9L and F98 cell lines), human

a

b


190

breast carcinoma (MCF-7), and human lung cancer (A-549, and NCI-H460).

The improvement in cytotoxicity has been attributed to the internalization

and intracellular drug delivery capacity of the nanocapsules in association

with a potential reversion of the multidrug resistance effect.

Importantly, the above indicated nanocapsules and those

functionalized with a monoclonal antibody (anti TMEFF-2) have also been

tested in a variety of animal models of cancer, such as: glyoma (Garcion et

al., 2006), hepatocellular carcinoma (Lacoeuille et al., 2007), colon

adenocarcinoma (Khalid et al., 2006), and lung cancer (Torrecilla et al.,

2011). The results obtained so far have shown an improved or similar

efficacy of the encapsulated drug as compared to that obtained for the free

drug. These are promising preliminary data as the nanocapsules are expected

to have a more acceptable toxicity profile (they avoid the use of toxic

solubilizers) than the commercial drugs.

Stability studies of HA nanocapsules during storage

Stability is a critical issue in the development of a nanocarrier

formulation. Variations on temperature are known to importantly

compromise the stability of colloidal systems, and show evident importance

during storage (Freitas and Müller, 1998). The size increase of the

formulations could be attributed to the weakening/breaking of linkers

between the molecules that forms the nanocarriers, and may alter their drug

release and induce destabilization of the formulations. A decrease in size

could also occur due to the detachment of components from the nanocarriers

or to an increase on the interaction strengths between linkers, potentially

affecting the desired efficacy of the nanocarrier. In the present study we

evaluated the stability of the DCX-loaded nanocapsules prepared with the

surfactant CTAB under storage at 4° and 37°C, for a period of 3 months.

Figure 5 shows that nanocapsules were generally stable during storage

Capitulo 4

191

irrespective of the temperature conditions. The overall trend of prolonged

stability is justified by the electrostatic repulsive effect due to the high zeta

potential values of nanocapsules (around -40 mV). Similar results were

obtained when the surfactant BKC was used for the elaboration of the

nanocapsules (data not shown). This acceptable stability profile has been

previously observed for other polymer nanocapsules (Prego et al., 2003;

Lozano et al. 2011b); and attributed to the polymer corona surrounding the

oily nanodroplets.

Figure 5: Stability study of DCX-loaded HA nanocapsules during 3 months of storage. Nanocapsules were prepared with CTAB and evaluated at 4°C ( ) and 37° C ( ). (Mean ± S.D.; n=3).

Formation of a lyophilized product of HA nanocapsules

Lyophilization is one of the most frequent and efficient methods for

preserving the properties of nanoparticulate systems during storage.

Nevertheless, this process becomes quite complex in the case of

nanocapsules due to the fluidity of the polymer shell and also due to the

presence of the oil core, which is susceptible of leakage (Choi et al., 2004). In

order to facilitate the lyophilization of the nanocarriers and avoid their

collapse, the use of cryoprotectant agents is necessary (Abdelwhaed et al.,


192

2006). The first evidence that polymeric nanocapsules were effectively

protected, by isotonic concentrations of cryoprotectants derived from sugar,

during a lyophilization process was carried out by Calvo et al. (1997).

Subsequent works (Lozano et al 2011a, b) have indicated that trehalose is an

adequate agent for preserving the stability of fluid nanosystems during

lyophilization. The main arguments that support the use of this agent over

others cryoprotectans is its less hygroscopicity together with its higher glass

transition temperature.

Figure 6 shows the size of HA nanocapsules containing BKC upon

reconstitution of the freeze-dried product. Overall the results indicate that at

relatively high concentrations of nanocapsules (1% w/v) it is possible to

achieve an adequate resuspension of the dried product without altering the

size of the nanocapsules.

Figure 6: Particle size of the reconstituted freeze dried HA nanocapsules using BKC as surfactant. Different concentrations (w/v) of nanocapsules were lyophilized using trehalose at 10% (black bars) or 5% w/v (grey bars). (Mean ± S.D.; n=3).

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193

Conclusions

In this paper we report the formation and characterization of a new

type of drug nanocarriers named as HA nanocapsules. Because of their

hydrophobic core, these nanocarriers are able to successfully encapsulate the

hydrophobic drug docetaxel. In addition, thanks to their HA hydrophilic

coating, these nanocarriers have the capacity to interact with NCI-H460

cancer cells and improve the pharmacological effect of the model drug

docetaxel. Finally, pharmaceutical parameters such as drug release, stability

during storage and reconstitution of freeze-dried HA nanocapsules render

these novel systems promising platforms for the intracellular delivery of

anticancer drugs.

References

1. Akima K, Ito H, Iwata Y et al.: Evaluation of antitumor activities of

hyaluronate binding antitumor drugs: synthesis, characterization and

antitumor activity. J. Drug Target 4,1-8 (1996).

2. Abdelwhaed W, Degobert G, Fessi H.: Investigation of nanocapsules

stabilization by amorphous excipients during freeze-drying and storage.

Eur. J. Pharm. Biopharm. 63, 87-94 (2006).

3. Ahmed IS, Ayres JW: Comparison of in vitro and in vivo performance

of a colonic delivery system. Int. J. Pharm. (2011),

doi:10.1016/j.ijpharm.2011.02.061.

4. Auzenne E, Ghosh SC, Khodadadian M et al.:. Hyaluronic acid-

paclitaxel: antitumor efficacy against CD44(+) human ovarian

carcinoma xenografts. Neoplasia 9, 479–486 (2007).

5. Bae KH, Lee Y, Park TG. Oil-encapsulating PEO-PPO-PEO/PEG shell

cross-linked nanocapsules for target-specific delivery of paclitaxel.

Biomacromol. 8(2), 650-6 (2007).


194

6. Calvo P, Remuñan-López C, Vila-Jato JL, Alonso MJ: Development of

positively charged colloidal drug carriers: Chitosan-coated polyester

nanocapsules and submicron-emulsions. Colloid. Polym. Sci. 275, 46-53

(1997).

7. Choi M.J, Briancon S, Andrieu J, Min SG, Fessi H: Effect of freeze-

drying process conditions on the stability of nanoparticles. Dry.

Technol. 22, 335-346 (2004).

8. Choi KY, Min KH, Na JH et al.: Self-assembled hyaluronic acid

nanoparticles as a potential drug carrier for cancer therapy: synthesis,

characterization, and in vivo biodistribution J. Mater. Chem. 19, 4102-

4107 (2009).

9. Crown J, O'Leary M: The taxanes: An update. Lancet 355(9210), 1176-

8 (2000).

10. Cowman M, Matsuoka SH: Experimental approaches to hyaluronan structure. Carbohydr Res. 340(5):791-809 (2005).

11. Eliaz RE, Szoka FCJr: Liposome-encapsulated doxorubicin targeted to

CD44: A strategy to kill CD44-overexpressing tumor cells. Cancer Res.

61(6), 2592-601 (2001).

12. Eliaz RE, Nir S, Marty C, Szoka FC Jr. Determination and modeling of

kinetics of cancer cell killing by doxorubicin and doxorubicin

encapsulated in targeted liposomes. Cancer Res. 64:711–718 (2004).

13. Freitas C, Müller RH: Effect of light and temperature on zeta potential

and physical and physical stability in solid lipid nanoparticle (SLNTM)

dispersions. Int. J. Pharm. 168, 221-229 (1998).

14. Garcion E, Lamprecht A, Heurtault B et al.: A new generation of

anticancer, drug-loaded, colloidal vectors reverses multidrug resistance

in glioma and reduces tumor progression in rats. Mol. Cancer Ther. 5(7),

1710-22 (2006).

Capitulo 4

195

15. Heurtault B, Saulnier P, Pech B, Proust JE, Benoit JP: A novel phase

inversion-based process for the preparation of lipid nanocarriers. Pharm.

Res. 19(6), 875-80 (2002a).

16. Heurtault B, Saulnier P, Pech B, Proust JE, Benoit JP: Properties of

polyethylene glycol 660 12-hydroxy stearate at a triglyceride/water

interface. Int. J. Pharm. 242(1-2), 167-70 (2002b).

17. Principles of Colloidal and Surface Chemistry (third edition). Hiemenz

PC, Rajagopalan R, (Editors). Dekker, New York, USA (1997).

18. Hyung W, Ko H, Park J et al.: Novel hyaluronic acid (HA) coated drug

carriers (HCDCs) for human breast cancer treatment. Biotechnol

Bioeng. 99(2), 442-54 (2008).

19. Khalid MN, Simard P, Hoarau D, Dragomir A, Leroux JC:. Long

circulating poly(ethylene glycol)-decorated lipid nanocapsules deliver

docetaxel to solid tumors. Pharm Res. 23(4), 752-8 (2006).

20. Lacoeuille F, Hindre F, Moal F et al.: In vivo evaluation of lipid

nanocapsules as a promising colloidal carrier for paclitaxel. Int. J.

Pharm. 344(1-2), 143-9 (2007).

21. Lee SH, Yoo SD, Lee KH: Rapid and sensitive determination of

paclitaxel in mouse plasma by high-performance liquid chromatography.

J. Chromatogr., B: Biomed. Sci. Appl., 724, 357-363 (1999).

22. Lozano MV, Torecilla D, Torres D, Vidal A, Dominguez F, Alonso MJ:

Highly efficient system to deliver taxanes into tumor cells: docetaxel-

loaded chitosan oligomer colloidal carriers. Biomacromol. 9, 2186-2193

(2008).

23. Lozano MV, Lollo G, Brea J, Torres D, Loza MI, Alonso MJ. Freeze-

dried polysaccharide nanocapsules: efficient vehicles for the

intracellular delivery of docetaxel. (submitted) (2011a).

24. Lozano MV, Lollo G, Brea J, Torres D, Loza MI, Alonso MJ.

Polyarginine nanocapsules: a new platform for intracellular drug

delivery. (submitted) (2011b).


196

25. Luo Y, Prestwich GD. Synthesis and selective cytotoxicity of a

hyaluronic acid-antitumor bioconjugate. Bioconjug. Chem. 10, 755–763

(1999).

26. Mossman T: Rapid colorimetric assay for cellular growth and survival:

application to proliferation and cytotoxicity assays. J. Immunol.

Methods 65, 55-63 (1983).

27. Ossipov DA: Nanostructured hyaluronic acid-based materials for active

delivery to cancer. Expert Opin. Drug Deliv. 7(6), 681-703 (2010).

28. Peer D, Margalit R: Tumor-targeted hyaluronan nanoliposomes increase

the antitumor activity of liposomal Doxorubicin in syngeneic and human

xenograft mouse tumor models. Neoplasia 6, 343-353 (2004a).

29. Peer D, Margalit R: Loading mitomycin C inside long circulating

hyaluronan targeted nano-liposomes increases its antitumor activity in

three mice tumor models. Int. J. Cancer 108, 780-789 (2004b).*

30. Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R:

Nanocarriers as an emerging platform for cancer therapy. Nat.

Nanotechnol. 2(12), 751-60 (2007).

31. Prego C, Fernandez-Megia E, Novoa-Carballal R, Quinoa E, Torres D,

Alonso MJ. Chitosan and chitosan-PEG nanocapsules: new carriers for

improving the oral absorption of calcitonin. Presented at: 30th Annual

Meeting of the Controlled Release Society, Glasgow, UK, 19-23 July

2003.

32. Prego C, García M, Torres D, Alonso MJ: Transmucosal

macromolecular drug delivery. J. Control Release 101(1-3), 151-62

(2005).

33. Prego C, Torres D, Alonso MJ: Chitosan nanocapsules: A new carrier

for nasal peptide delivery. JDDST. 16, 331-337 (2006).

34. Rosato A, Banzato A, De Luca G et al.: HYTAD1-p20: a new

paclitaxel-hyaluronic acid hydrosoluble bioconjugate for treatment of

superficial bladder cancer. Urol. Oncol. 3, 207-15 (2006).

Capitulo 4

197

35. Handbook of pharmaceutical excipients (Sixth edition). Raymond C

Rowe, Paul J Sheskey, Marian E Quinn (Editors). Pharmaceutical press,

London-United Kingdom (2009).

36. Santander-Ortega MJ, Peula-García JM, Goycoolea FM, Ortega-

Vinuesa JL: Chitosan nanocapsules: Effect of chitosan molecular weight

and acetylation degree on electrokinetic behaviour and colloidal

stability. Colloids Surf. B Biointerfaces. 82(2), 571-80 (2011).

37. Surace C, Arpicco S, Dufaÿ-Wojcicki A et al.: Lipoplexes targeting the

CD44 hyaluronic acid receptor for efficient transfection of breast cancer

cells. Mol Pharm. 6(4), 1062-73 (2009).

38. Ten Tije AJ, Verweij J, Loos WJ, Sparreboom A: Pharmacological

effects of formulation vehicles : implications for cancer chemotherapy.

Clin. Pharmacokinet. 42(7), 665-85 (2003).

39. Torrecilla D, Lozano MV, Lallana E: In Vivo Efficacy of Anti-TMEFF-

2 Modified Chitosan-g-Poly(ethyleneglycol) Nanocapsules in Non-

Small Cell Lung Tumors (submitted) (2011).

Discusión

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DISCUSIÓN

El ácido hialurónico (HA) es un polísacárido aniónico natural, no

tóxico y biodegradable que se ha revelado como una propuesta muy

interesante para el diseño de nanosistemas dirigidos específicamente al

tratamiento de tumores sólidos118;119;120;121. Este polímero posee especificidad

frente a los receptores CD-44 sobreexpresados en diferentes tipos de cáncer,

entre otros los que cursan a nivel pulmonar como el cáncer de células no

pequeñas, metaplasia escamosa y adenocarcinoma122;123.

El HA posee además una demostrada capacidad mucoadhesiva 124;125

que lo posiciona como un material ideal para dotar a los nanosistemas de la

mucoadhesión necesaria para favorecer el contacto con el epitelio pulmonar y

potenciar la difusión de fármacos antitumorales hacia el entorno tumoral y su

penetración intracelular.

Los nanosistemas que contienen un núcleo oleoso contituyen el

vehículo ideal para fármacos antitumorales extremadamente hidrofóbicos,

como son los taxanos126;127;128;129. Concretamente, el docetaxel ha sido

incluído en el núcleo oleoso de sistemas nanométricos como

118 Surace C, Arpicco S, Dufaÿ-Wojcicki A, Marsaud V, Bouclier C, Clay D, Cattel L, Renoir JM, Fattal E. (2009). Mol Pharm. 6(4):1062-73. 119 Taetz S, Bochot A, Surace C, Arpicco S, Renoir JM, Schaefer UF, Marsaud V, Kerdine-Roemer S, Lehr CM, Fattal E. (2009). Oligonucleotides. 19(2):103-16. 120 Peer D, Margalit R. (2004). Neoplasia. 6(4):343-53. 121 Eliaz RE, Nir S, Szoka FC Jr. (2004) Methods Enzymol. 387:16-33. 122 Penno MB, August JT, Baylin SB, Mabry M, Linnoila RI, Lee VS, Croteau D, Yang XL, Rosada C. (1994). Cancer Res. 54(5):1381-7. 123 Tran TA, Kallakury BV, Sheehan CE, Ross J.S. (1997). Hum Pathol. 28(7):809-14. 124 Di Colo G, Zambito Y, Zaino C, Sansò M. (2009). Drug Dev Ind Pharm. 35(8):941-9. 125 Saso L, Bonanni G, Grippa E, Gatto MT, Leone MG, Silvestrini B. (1999). Res Commun Mol Pathol Pharmacol. 104(3):277-84. 126 Huynh N.T.; Passirani C, Saulnier P, Benoit J.P. (2009). Int. J. Pharm. 379(2):201-9. 127 Bae K.H.; Lee Y.; Park T.G. (2007). Biomacromolecules. 8: 650-656. 128 Khalid M.N.; Simard P.; Hoarau D.; Dragomir A.; Leroux J.C. (2007). Pharm. Res. 23: 752-758. 129 Peltier S, Oger JM, Lagarce F, Couet W, Benoit J.P. (2006). Pharm. Res. 23: 1243-1250.

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nanocápsulas126;128;130;131 y liposomas132;133;134;135;136;137, lo que ha permitido

con mayor o menor éxito potenciar su efecto antitumoral, evitando la

utilización de los disolventes incluidos hasta ahora en la formulación

comercial.

En nuestro grupo de investigación, la inclusión de docetaxel en

nanocápsulas de quitosano131 y poliarginina138, ha confirmado la idoneidad de

estos nanosistemas en cuanto a lograr una encapsulación adecuada del

fármaco, y potenciar su vehiculización hacia el interior de las células

tumorales.

El trabajo central de la segunda parte de esta tesis se ha dirigido a

explorar un nuevo nanosistema, las nanocápsulas de HA, como vehículos del

antitumoral docetaxel, con el fin último de lograr una aplicación localizada

pulmonar.

Preparación y caracterización de las nanocápsulas de HA

Las nanocápsulas de HA se prepararon mediante la la técnica de

desplazamiento de disolvente, adaptando un procedimiento desarrollado

previamente en nuestro laboratorio para otros polímeros catiónicos, como el

130 Nassar T, Attili-Qadri S, Harush-Frenkel O, Farber S, Lecht S, Lazarovici P, Benita S. (2011). Cancer Res. 71(8):3018-28. 131 Lozano MV, Torrecilla D, Torres D, Vidal A, Domínguez F, Alonso MJ. (2008) Biomacromolecules. 9(8):2186-93. 132 Immordino ML, Brusa P, Arpicco S, Stella B, Dosio F, Cattel L. (2003). J. Control Release. 91(3):417-29. 133 Qin Y, Song QG, Zhang ZR, Liu J, Fu Y, He Q, Liu J. (2008) Arzneimittelforschung. 58(10):529-34. 134 Liang G, Jia-Bi Z, Fei X, Bin N. (2007). J. Pharm. Pharmacol. 59(5):661-7. 135 Yuan Z, Chen D, Zhang S, Zheng Z. (2010). Yakugaku Zasshi. 130(10):1353-9. 136 Yamamoto Y, Yoshida M, Sato M, Sato K, Kikuchi S, Sugishita H, Kuwabara J, Matsuno Y, Kojima Y, Morimoto M, Horiuchi A, Watanabe Y. (2011). Int. J. Oncol. 38(1):33-9. 137 Zhai G, Wu J, Yu B, Guo C, Yang X, Lee R.J. (2010). J. Nanosci. Nanotechnol. 10(8):5129-36. 138 Lozano MV, Lollo G, Brea J, Loza MI, Torres D, Alonso MJ. (2011). Submitted.

Discusión

201

quitosano131;139 y la poliarginina138. En el caso de los polímeros catiónicos, la

formación de nanocápsulas se producía gracias a la interacción de éstos con

el tensoactivo aniónico, lecitina, incluido en el núcleo de una nanoemulsión.

Para la formación de las nanocápsulas de HA, al tratarse de un polímero

aniónico, fue necesario adaptar el método, mediante la incorporación de un

tensoactivo catiónico. Básicamente, el proceso consistió en la adición, bajo

agitación suave, de una fase orgánica etanol-acetona (conteniendo el aceite

Mygliol 812®, lecitina y el tensoactivo catiónico) sobre una fase acuosa

conteniendo poloxámero 188, tras lo cual se obtiene la nanoemulsión

catiónica (Figura 1).

Figura 1. Esquema de preparación de las nanocápsulas de HA

Una vez eliminados los solventes en rotavapor, la nanoemulsión se

incubó con una solución acuosa del HA en una relación de volúmenes 4:1.5.

En estas condiciones, el HA se dispone, a través de interacciones

electrostáticas, sobre la superficie de la nanoemulsión, originandose las

nanocápsulas. Una estrategia similar, pero orientada a la formación de

nanopartículas de poli-ε-caprolactona recubiertas con HA, ha sido

previamente descrita por Barbault-Foucher y col140.

Es importante señalar que las nanocápsulas se pueden formar

igualmente en una sola etapa, es decir añadiendo directamente la fase

139 Prego C, García M, Torres D, Alonso MJ. (2005). J Control Release. 101(1-3):151-62. 140 Barbault-Foucher S, Gref R, Russo P, Guechot J, Bochot A. (2002). J. Control Release. 83(3):365-75.

Discusión

202

orgánica sobre la fase acuosa conteniendo el polímero, dando lugar a

resultados muy similares los discutidos en esta parte del trabajo (datos no

mostrados).

Para la obtención de nanocáspulas estables, se ha evaluado el efecto

de tres factores sobre el proceso de formación: tipo de tensoactivo catiónico,

peso molecular y concentración de HA. Los tensoactivos catiónicos

seleccionados para el estudio fueron el cloruro de benzalconio (BKC) y el

bromuro de hexadeciltrimetilamonio (CTAB), los pesos moleculares de HA

estudiados fueron bajo (29 KDa) y alto (160 KDa) peso molecular, y la

concentración de polímero se evaluó a 8 y 9 niveles (Tablas 1 y 2).

Las cantidades de BKC y CTAB utilizadas en la preparación de las

formulaciones fueron 4 y 1.8 mg, respectivamente. El criterio de selección

de estas cantidades obedece a que fueron las mínimas necesarias para lograr

nanoemulsiones catiónicas estables.

El efecto de la concentración de HA sobre el tamaño y carga

superficial de las nanocápsulas formadas se muestra en las Tablas 1a y 1b,

que corresponden a los tensoactivos BKC y CTAB, respectivamente. La

incubación de las nanoemulsiones catiónicas con concentraciones adecuadas

de HA permite la formación de poblaciones homogéneas de nanocápsulas

con un tamaño medio aproximado de 250 nm. Este tamaño coincide con el

presentado por nanocápsulas recubiertas con quitosano131;139 y

poliarginina138, preparadas por un método similar. Mediante microscopía

electrónica de transmisión (Figura 2) se confirmó la esfericidad de estas

nanocápsulas, que muestran la estructura núcleo-cubierta similar a otros

sistemas nanocapsulares de diferente cubierta131;138.

En los valores de la carga superficial, se puede apreciar que, a

medida que se aumenta la cantidad de HA, se presenta un enmascaramiento

parcial o una completa inversión del potencial zeta de las nanoemulsiones

Discusión

203

catiónicas originales, lo que sugiere que el polímero cargado negativamente

se dispone en la superficie de los nanosistemas.

Tabla 1. Características físico-químicas de las nanocápsulas preparadas con distintas cantidades de HA (29 kDa), utilizando como tensoactivos (a) BKC o (b) CTAB (media ± d.e., n≥3).

HA (mg) Tamaño (nm) Índice de polidispersión Potencial ζ (mV) 0 223 ± 10 0.1 27 ± 3

0.5 249 ± 10 0.2 9 ± 2 1.0 pp. --- --- 3.1 pp. --- --- 6.2 248 ± 10 0.2 -42 ± 2

12.5 235 ± 13 0.1 -45 ± 4 25.0 252 ± 19 0.1 -46 ± 2 50.0 pp. --- ---

HA (mg) Tamaño (nm) Índice de polidispersión Potencial ζ (mV) 0 215 ± 7 0.1 35 ± 2

0.1 231 ± 8 0.1 18 ± 2 0.5 pp. --- --- 1.0 pp. --- --- 3.1 238 ± 2 0.1 -35 ± 2 6.2 238 ± 3 0.1 -37 ± 2

12.5 260 ± 20 0.2 -31 ± 3 25.0 pp. --- --- 50.0 pp. --- ---

pp.= precipitación

En las Tablas 1a y 1b se pueden apreciar 2 puntos críticos en los que

ocurre la precipitación de los nanosistemas. Uno de ellos está bastante

cercano a la zona en la que se produce la inversión de la carga superficial. En

este caso, la precipitación ocurre, presumiblemente, debido a que la cantidad

de HA no es suficiente para provocar la inversión del potencial zeta de los

sistemas, favoreciendo únicamente la contraionización de la carga positiva de

superficie. Esta contraionización provoca que el potencial zeta de las

formulaciones se acerque a la neutralidad, impidiendo la repulsión eléctrica

entre los nanosistemas, y ocasionando su precipitación.

(a)

(b)

Discusión

204

Figura 2. Imágenes de microscopía electrónica de transmisión de nanocápsulas obtenidas con 12.5 mg of HA (29 kDa).

El otro punto crítico en el que se observa precipitación se relaciona

con el exceso de HA. Una combinación de factores como son la mayor fuerza

iónica141 (el HA está en forma de sal sódica) y la mayor viscosidad del

medio, es lo que provocaría la precipitación de los nanosistemas142.

Los valores correspondientes a tamaño, polidispersión y potencial

zeta de las formulaciones preparadas con el HA de mayor peso molecular

(Capítulo 4, Tabla 3) resultaron superiores a los obtenidos con el HA de

bajo peso molecular Esta diferencia se atribuye a que la adsorción superficial

del polímero de alto peso molecular produce un aumento en el grosor de la

cubierta, lo que conlleva a un aumento en el tamaño de los nanosistemas.

Este mayor grosor en la cubierta se asocia a un mayor número de grupos

ionizables en superficie, lo que explicaría el aumento en el potencial zeta.

Considerando esta información, se eligió el HA de peso molecular 29 KDa

para continuar los experimentos de encapsulación del docetaxel y eficacia de

los nanosistemas.

141 Hiemenz PC, Rajagopalan R. (1997). Principles of Colloidal and Surface Chemistry. Dekker, New York. 142 Cowman M, Matsuoka SH. (2005). Carbohydrates res. 340(5):791-809.

Discusión

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Encapsulación y liberación de docetaxel a partir de las nanocápsulas

Para llevar a cabo la encapsulación del antitumoral, hemos

seleccionado las formulaciones elaboradas con 12.5 mg de HA. El docetaxel

se encapsuló eficazmente (Tabla 2), sin que se produjesen modificaciones

significativas en las características físico-químicas originales de las

nanocápsulas. Estos datos coinciden con los mostrados en trabajos publicados

previamente, y se atribuyen a la afinidad del fármaco por establecer

interacciones hidrofóbicas con los componentes del núcleo de las

nanocápsulas128;131;138.

Tabla 2. Características físico-químicas y eficacia de encapsulación de las formulaciones de nanocápsulas de HA conteniendo docetaxel (DCX), elaboradas con los tensoactivos BKC y CTAB (media ± d.e., n≥3).

Formulación Tamaño (nm)

Indice de polidispersión

Potencial ζ (mV)

Eficacia de encapsulación

(%) Nanocápsulas blancas de HA

(BKC) 235 ± 13 0.1 -45 ± 4 -

Nanocápsulas de HA-DCX(BKC) 250 ± 20 0.1 -52 ± 11 65 ± 3

Nanocápsulas blancas de HA

(CTAB) 267 ± 23 0.1 -31 ± 3 -

Nanocápsulas de HA-CX (CTAB) 276 ± 1 0.1 -36 ± 1 65 ± 3

La liberación de docetaxel a partir de las nanocápsulas muestra una

evolución similar para ambos tensoactivos utilizados. El perfil es bifásico,

caracterizado por una rápida liberación inicial seguida por una segunda etapa

de estabilización. La etapa de liberación inicial, que es típica de este tipo de

sistemas de naturaleza oleosa128;131, se relaciona con la dilución de las

nanocápsulas en el medio de incubación y el consiguiente reparto del

fármaco entre el núcleo oleoso y el medio externo. La ausencia de liberación

en la segunda etapa confirma la alta afinidad que posee el docetaxel para

interaccionar con los componentes oleosos de los nanosistemas.

Discusión

206

Figura 2: Perfiles de liberación in vitro de docetaxel a partir de las nanocápsulas de HA elaboradas con los tensoactivos BKC (X) o CTAB ( ) (media± d.e., n=3).

En términos cuantitativos, la liberación del fármaco se muestra muy

similar a la observada con nanocápsulas de quitosano131 y poliarginina138, lo

que indica que la composición de la cubierta polimérica (HA, quitosano o

poliarginina) no tiene una influencia significativa sobre la liberación del

fármaco.

Eficacia antitumoral de las nanocápsulas de HA conteniendo docetaxel

sobre la línea celular NCI-H460

Los estudios de viabilidad celular se realizaron en una línea de cáncer

de pulmón que sobreexpresa el receptor CD-44 (NCI-H460). Este receptor se

expresa de manera importante en tumores sólidos143; de tal manera que se

utiliza como marcador celular para identificar el cáncer de células no

pequeñas de pulmón, metaplasia escamosa y adenocarcinoma89;90.

Los resultados de viabilidad celular, tras un contacto de 48 h con las

nanocápsulas conteniendo docetaxel, o sus controles (Figuras 3a y 3b),

143 Naor D, Nedvetski S, Golan L, Melnik L, Faitelson Y. (2002) CD44. Crit. Rev. Clin. Lab. Sci. 39(6):527-79.

Discusión

207

indican una clara potenciación del efecto antitumoral del docetaxel, tras su

inclusión en los nanosistemas. Este efecto resulta significativo para todas las

concentraciones de fármaco ensayadas, en el caso de las nanocápsulas

preparadas con el tensoactivo CTAB, mientras que con el BKC, lo es a partir

de 12.5 nM de fármaco.

Los valores de la concentración de fármaco necesaria para producir el

50% de la muerte celular, IC50 (Tabla 4), confirman el incremento de la

eficacia antitumoral logrado cuando el fármaco se incorpora a las

nanocápsulas (3.4 y 2.4 veces más efectivas que el docetaxel en solución,

para las elaboradas con CTAB y BCK, respectivamente).

En el caso de las nanocápsulas blancas elaboradas con BCK, se

observa para las concentraciones más altas una cierta reducción de la

viabilidad, atribuible al vehículo por si mismo. Este efecto se debe relacionar

con la mayor toxicidad del BCK, en comparación con el CTAB144, y con el

hecho de que se necesita una mayor concentración del primero para conseguir

formulaciones estables.

a

0102030405060708090100110

6,25 12,5 25 50 75

Via

bilid

ad c

elul

ar (%

)

Docetaxel (nM)

Nanocápsulas blancas de HA (CTAB)

DCX en solución

Nanocápsulas de HA (CTAB) cargadas con DCX

###

#

#

144 Rowe RC, Sheskey PJ, Quinn, ed. Handbook of pharmaceutical excipients, 6th edn. United Kingdom: pharmaceutical press, 2009.

Discusión

208

b

0

10

20

30

40

50

60

70

80

90

100

110

6,25 12,5 25 50 75

Via

bilid

ad c

elul

ar (%

)

Docetaxel (nM)

Nanocápsulas blancas de HA (BKC)

DCX en solución

Nanocápsulas de HA (BKC) cargadas con DCX

## #

#

Figura 3: Efecto de las nanocápsulas de HA blancas (blanco), docetaxel (DCX) en solución (gris) y y nanocápsulas de HA conteniendo DCX (negro) sobre la viabilidad de células NCI-H460, tras 48 h de incubación, utilizando el tensoactivo CTAB (a) o BKC (b). #Diferencias significativas entre nanocápsulas conteniendo DCX vs. DCX en solución (p<0.005). Tabla 4. Valores de IC50 (nM ;n=4), obtenidos tras el contacto de las formulaciones indicadas con células NCI-H460, durante un período de exposición de 48 h.

Formulación IC50 (nM) Nanocápsulas blancas de HA (CTAB) n.d. Nanocápsulas blancas de HA (BKC) *

DCX en solución 36.4±4.0 Nanocápsulas de HA (CTAB) conteniendo DCX 10.8±1.1# Nanocápsulas de HA (BKC) conteniendo DCX 15.3±2.0#

*: La máxima reducción en viabilidad celular fue de 39.0 ± 8.0%. #: P<0.01 con respecto a la IC50 de DCX (One-way ANOVA test, post-hoc Tukey test).

Los resultados de la viabilidad celular tras un contacto de 2 h con las

formulaciones (Capítulo IV, Figuras 3a y 4a), indicaron cómo ya tras un

período mínimo de contacto, el efecto del docetaxel encapsulado es

marcadamente superior al del docetaxel en solución, lo que apunta a que las

nanocápsulas favorecen la penetración intracelular del fármaco.

Discusión

209

La significativa mejora de la eficacia antitumoral del docetaxel

cuando se incorpora a las nanocápsulas de HA es concordante con lo

observado en otros estudios en los que nanocápsulas cargadas con taxanos y

recubiertas con PEG145, quitosano131 o poliarginina138, se evaluaron en líneas

celulares de glioma (9L y F98), carcinoma de mama (MCF-7) y cáncer de

pulmón (A549 y NCI-H460), respectivamente. En el caso de las

formulaciones recubiertas con PEG, la mejora se atribuyó a la capacidad de

las nanoestructuras para contrarrestar el efecto de resistencia múltiple a

fármacos. Para los nanosistemas de quitosano y poliarginina, el efecto se

relacionó con la capacidad de los polímeros cargados positivamente para

conseguir una mejor interacción/internalización de los nanosistemas. Estas

formulaciones fueron también ensayadas in vivo en modelos animales a los

que se implantaron subcutáneamente células de glioma145, carcinoma

hepatocelular146, adenocarcinoma de colon128 y cáncer de pulmón147. Los

resultados de estos experimentos mostraron una eficacia similar o superior a

la del fármaco sólo pero, a diferencia de lo que sucede con éste (formulación

comercializada), los nanosistemas estudiados no necesitan ser administrados

con solventes y tensoactivos que solubilizan el fármaco, a costa de producir

importantes efectos de sensibilización y toxicidad.

Los resultados presentados son prometedores, y hacen esperar un

mejor resultado tras la utilización de los nanosistemas de HA por vía

inhalatoria, para el tratamiento localizado de cáncer de pulmón, ya que

permitiría potenciar aun más la eficacia del antitumoral, disminuyendo la

dosis utilizada por vía endovenosa148.

145 Garcion E, Lamprecht A, Heurtault B, Paillard A, Aubert-Pouessel A, Denizot B, Menei P, Benoît JP. (2006). Mol. Cancer Ther. 5(7):1710-22. 146 Lacoeuille F, Hindre F, Moal F, Roux J, Passirani C, Couturier O, Cales P, Le Jeune JJ, Lamprecht A, Benoit JP. (2007). Int J Pharm. 344(1-2):143-9. 147 Torrecilla D, Lozano MV, Lallana E, Novoa-Carballal R, Vidal A, Torres D, Fernández-Megía E, Riguera E, Alonso MJ, Domínguez F. (2011). Sometido. 148 Hureaux J, Lagarce F, Gagnadoux F, Vecellio L, Clavreul A, Roger E, Kempf M, Racineux JL, Diot P, Benoit JP, Urban T. (2009). Eur J Pharm Biopharm. 73(2):239-46.

Discusión

210

Estudios de estabilidad en almacenamiento

de las nanocápsulas de HA conteniendo docetaxel

La inestabilidad es un problema crítico frecuente en el desarrollo

de formulaciones de sistemas coloidales. En el presente estudio, se ha

evaluado la estabilidad de las formulaciones de nanocápsulas de HA con

docetaxel, en suspensión, almacenadas a 4 y 37° C, durante un periodo de 3

meses. En la Figura 5 se puede apreciar que todas las formulaciones se

mantienen estables, en lo que se refiere al tamaño, durante el periodo de

estudio. Esta prolongada estabilidad puede ser atribuida al potencial zeta

altamente negativo que presentan los sistemas durante todo el experimento (~

-40 mV). Estos resultados concuerdan con los encontrados en otros sistemas

nanocapsulares149 en los que la estabilidad coloidal se ve favorecida por la

presencia de la cubierta polimérica.

Figura 5: Evolución del tamaño de particula de las nanocápsulas de HA (tensoactivo CTAB) conteniendo docetaxel durante 3 meses de almacenamiento a 4 ( ) y 37° C ( ). (Media ± d.e.; n=3).

149 Prego C, Torres D, Fernandez-Megia E, Novoa-Carballal R, Quiñoá E, Alonso MJ. J Control Release. (2006). 111(3):299-308.

Discusión

211

Obtención de un producto liofilizado de nanocápsulas de HA

Una de las limitaciones tecnológicas comunes a los nanosistemas es

su deficiente estabilidad durante largos periodos de tiempo. La gelificación,

cremación, fusión o agregación son los fenómenos más comunes de

desestabilización que pueden sufrir estos sistemas durante su

almacenamiento150.

La liofilización es uno de los métodos más eficaces para preservar su

estabilidad durante largos periodos de tiempo. Sin embargo, este proceso se

puede tornar especialmente complejo en el caso de las nanocápsulas debido a

la fluidez de la cubierta polimérica y a la presencia del núcleo oleoso que es

susceptible de romperse151.

El uso de agentes crioprotectores durante la liofilización de las

nanoestructuras es fundamental pues facilita el proceso y previene el colapso

de los nanosistemas152. La primera evidencia de que concentraciones

isótónicas de criprotectores derivados de azúcares son capaces de proteger a

las nanocápsulas durante su liofilización fue aportada por Calvo y col.

(1997)153. Estudios posteriores llevados a cabo con diferentes estructuras

como nanopartículas, liposomas y nanocápsulas han indicado que la trealosa

es uno de los agentes más eficaces para asegurar un proceso de liofilización y

reconstitución adecuada de los nanosistemas (su menor higroscopicidad y su

mayor temperatura de transición vítrea son las características que la

diferencian de otros azúcares).

150 Heurtault B, Saulnier P, Pech B, Proust JE, Benoit JP. (2003). Biomaterials. 24:4283-4300. 151 Choi MJ, Briancon S, Andrieu J, Min SG, Fessi H. (2004). Dry. Technol. 22:335-346. 152 Abdelwhaed W.; Degobert G.; Fessi H. (2006). Eur. J. Pharm. Biopharm. 63:87-94. 153 Calvo P, Remuñan-López C, Vila-Jato JL, Alonso MJ. (1997). Colloid. Polym. Sci. 275, 46-53.

Discusión

212

Los resultados obtenidos con las nanocápsulas de HA (Figura 6) nos

llevan a concluir que la trealosa es un agente que permite obtener un

producto liofilizado adecuado, llegando a concentraciones del nanosistema

del 1% p/v.

Figura 6: Tamaño de partícula de las nanocápsulas de HA conteniendo docetaxel (tensoactivo: BKC) tras el proceso de liofilización y reconstitución de la suspensión Las nanocápsulas se liofilizaron en presencia de trealosa al 5 y 10 % . (media ± d.e.; n=3).

CONCLUSIONES

Conclusiones

215

CONCLUSIONES

El trabajo experimental expuesto pone de manifiesto el interés de las

nanostructuras desarrolladas para cumplir el objetivo futuro de una

administración pulmonar eficaz. Ello se ha podido constatar a través del

estudio de la interacción de las nanoestructuras con las células diana

(mastocitos y células de cáncer de pulmón) y de la cuantificación de la

eficacia antiasmática y antitumoral de los fármacos asociados.

.

De todo ello, se han podido extraer las siguientes conclusiones:

PARTE I

1. Se han optimizado, utilizando el método de gelificación iónica,

sistemas nanoparticulares constituídos por mezclas quitosano-ácido

hialurónico y quitosano-carboximetil-β-ciclodextrina, con un elevado

contenido en heparina.

2. El tamaño medio osciló en torno a 150-375 nm, dependiendo

fundamentalmente del peso molecular de la heparina asociada, mostrando

siempre una elevada carga superficial positiva. Los perfiles de estabilidad de

los nanosistemas y de liberación de heparina de alto y bajo peso molecular,

se mostraron muy dependientes de la composición de las formulaciones y

medios utilizados.

3. Se evidenció, mediante microscopía confocal, que las nanopartículas,

independientemente de su composición, fueron internalizadas por los

mastocitos, demostrándose ex vivo, por primera vez para un sistema

nanoparticular, y concretamente para las nanopartículas de quitosano-

Conclusiones

216

ciclodextrina, la mejora del efecto antiasmático de la heparina, frente al de la

molécula en solución.

PARTE II

1. Se ha diseñado, mediante la técnica de desplazamiento del

disolvente, un nuevo sistema que denominamos “nanocápsulas de ácido

hialurónico”, constituído por un núcleo oleoso que contiene el principio

activo y una cubierta de ácido hialurónico.

2. Los nanosistemas presentaron tamaño nanométrico (en torno a 250

nm) y carga superficial negativa (entre -30 y -50 mV), mostrando la

capacidad de encapsular eficazmente el antitumoral hidrofóbico docetaxel y

retenerlo en el núcleo oleoso tras su dilución en fluídos biológicos simulados

3. El docetaxel encapsulado incrementó significativamente su efecto

citotóxico sobre células tumorales de cáncer de pulmón (NCI-H460),

incremento que se atribuye a la internalización de las nanocápsulas y

posterior liberación intracelular del docetaxel.

4. Las nanocápsulas almacenadas en forma de suspensión fueron

estables durante al menos un período de 3 meses, independientemente de la

temperatura de almacenamiento (4 ó 37° C).

5. Las nanocápsulas se pudieron liofilizar y reconstituir adecuadamente,

tras la incorporación del crioprotector trealosa en concentraciones en torno al

5% p/v.

REFERENCIAS

Referencias

219

REFERENCIAS

1. Abal M, Andreu JM, Barasoain I. Taxanes: microtubule and centrosome

targets, and cell cycle dependent mechanisms of action. Curr Cancer

Drug Targets. 3(3):193-203 (2003).

2. Abdelwahed W, Degobert G, Fessi H. Investigation of nanocapsules

stabilization by amorphous excipients during freeze-drying and storage.

Eur J Pharm Biopharm. 63(2):87-94 (2006).

3. Aboubakar M, Puisieux F, Couvreur P, Deyme M, Vauthier C. Study of

the mechanism of insulin encapsulation in poly(isobutylcyanoacrylate)

nanocapsules obtained by interfacial polymerization. J Biomed Mater

Res. 47:568-576 (1999).

4. Ahmad Z, Sharma S, Khuller GK. Inhalable alginate nanoparticles as

antitubercular drug carriers against experimental tuberculosis. Int. J.

Antimicrob. Agents. 26(4):298-303 (2005).

5. Ahmed IS, Ayres JW: Comparison of in vitro and in vivo performance

of a colonic delivery system. Int. J. Pharm. (2011),

doi:10.1016/j.ijpharm.2011.02.061.

6. Ahmed T, Campo C, Abraham MK, Molinari JF, Abraham WM, Ashkin

D, Syriste T, Andersson LO, Svahn CM. Inhibition of antigen-induced

acute bronchoconstriction, airway hyperresponsiveness, and mast cell

degranulation by a nonanticoagulant heparin: comparison with a low

molecular weight heparin. Am J Respir Crit Care Med. 155(6):1848-55

(1997).

7. Ahmed T, Garrigo J, Danta I. Preventing bronchoconstriction in

exercise-induced asthma with inhaled heparin. N Engl J Med.

329(2):90-5 (1993).

Referencias

220

8. Ahmed T, Gonzalez BJ, Danta I. Prevention of exercise-

induced bronchoconstriction by inhaled low-molecular-weight heparin.

Am J Respir Crit Care Med. 160(2):576-81 (1999).

9. Ahmed T, Ungo J, Zhou M, Campo C. Inhibition of allergic late airway

responses by inhaled heparin-derived oligosaccharides. J Appl Physiol.

88(5):1721-9 (2000).

10. Ahmed T, Syriste T, Mendelssohn R, Sorace D, Mansour E, Lansing

M, Abraham WM, Robinson MJ. Heparin prevents antigen-induced

airway hyperresponsiveness: interference with IP3-mediated mast cell

degranulation? J Appl Physiol. 76(2):893-901 (1994).

11. Al Khouri Fallouh N, Roblot-Treupel L, Fessi H. Development of a new

process for the manufacture of polyisobutylcyanoacrylate nanocapsules.

Int J Pharm. 28:125-132 (1986).

12. Akima K, Ito H, Iwata Y, Matsuo K, Watari N, Yanagi M, Hagi

H, Oshima K, Yagita A, Atomi Y, Tatekawa I. Evaluation of antitumor

activities of hyaluronate binding antitumor drugs: synthesis,

characterization and antitumor activity. J Drug Target. 4(1):1-8 (1996).

13. Alonso MJ. Nanomedicines for overcoming biological barriers. Biomed

Pharmacother. 58:168-172 (2004).

14. Alonso MJ, Sanchez, A. The potential of chitosan in ocular drug

delivery. J Pharm Pharmacol. 55:1451-1463 (2003).

15. Andrade F, Videira M, Ferreira D, Sarmento B. Nanocarriers for

pulmonary administration of peptides and therapeutic proteins.

Nanomedicine (Lond). 6(1):123-41 (2011).

16. Anton N, Gayet P, Benoit JP, Saulnier P. Nano-emulsions and

nanocapsules by the PIT method: An investigation on the role of the

temperature cycling on the emulsion phase inversion. Int J Pharm.

344:44-52 (2007).

Referencias

221

17. Aspden TJ, Mason JD, Jones NS, Lowe J, Skaugrud O, Illum L.

Chitosan as a nasal delivery system: the effect of chitosan solutions on

in vitro and in vivo mucociliary transport rates in human turbinates and

volunteers. J. Pharm Sci. 86(4):509-13 (1997).

18. Auzenne E, Ghosh SC, Khodadadian M, Rivera B, Farquhar D, Price

RE, Ravoori M, Kundra V, Freedman RS, Klostergaard J. Hyaluronic

acid-paclitaxel: antitumor efficacy against CD44(+) human ovarian

carcinoma xenografts. Neoplasia. 9(6):479-86 (2007).

19. Bae KH, Lee Y, Park TG. Oil-encapsulating PEO-PPO-PEO/PEG shell

cross-linked nanocapsules for target-specific delivery of paclitaxel.

Biomacromolecules. 8(2):650-6 (2007).

20. Barar J, Javadzadeh AR, Omidi, Y. Ocular novel drug delivery: Impacts

of membranes and barriers. Expert Opin Drug Deliv. 5:567-581 (2008).

21. Barbault-Foucher S, Gref R, Russo P, Guechot J, Bochot A. Design of

poly-epsilon-caprolactone nanospheres coated with bioadhesive

hyaluronic acid for ocular delivery. J Control Release. 83(3):365-75

(2002).

22. Barnes PJ. New therapies for asthma: is there any progress? Trends

Pharmacol. Sci. 31(7):335-43 (2010).

23. Becquemin MH, Chaumuzeau JP. Inhaled insulin: a model for

pulmonary systemic absorption? Rev. Mal. Respir. 27(8):e54-65 (2010).

24. Bivas-Benita M, Ottenhoff TH, Junginger HE, Borchard G.

Pulmonary DNA vaccination: concepts, possibilities and

perspectives. J Control Release. 107(1):1-29 (2005).

25. Bivas-Benita M, Romeijn S, Junginger HE, Borchard G. PLGA-PEI

nanoparticles for gene delivery to pulmonary epithelium. Eur J Pharm

Biopharm. 58(1):1-6 (2004).

Referencias

222

26. Bivas-Benita M, van Meijgaarden KE, Franken KL, Junginger

HE, Borchard G, Ottenhoff TH, Geluk A.Pulmonary delivery of

chitosan-DNA nanoparticles enhances the immunogenicity of a DNA

vaccine encoding HLA-A*0201-restricted T-cell epitopes of

Mycobacterium tuberculosis. Vaccine. 22(13-14):1609-15 (2004).

27. Bivas-Benita M, Zwier R, Junginger HE, Borchard G. Non-invasive

pulmonary aerosol delivery in mice by the endotracheal route. Eur J

Pharm Biopharm. 61(3):214-8 (2005).

28. Bouclier Cl, Moine L, Hillaireau H, Marsaud VR, Connault E, Opolon

P, Couvreur P, Fattal E, Renoir JM. Physicochemical Characteristics

and Preliminary in Vivo Biological Evaluation of Nanocapsules Loaded

with siRNA Targeting Estrogen Receptor Alpha. Biomacromolecules.

9:2881-2890 (2008).

29. Brigger I, Dubernet C, Couvreur P. Nanoparticles in cancer therapy and

diagnosis. Adv Drug Deliv Rev. 54:631-651 (2002).

30. Brown SD, Nativo P, Smith JA, Stirling D, Edwards PR, Venugopal

B, Flint DJ, Plumb JA, Graham D, Wheate NJ. Gold nanoparticles for

the improved anticancer drug delivery of the active component of

oxaliplatin. J Am Chem Soc. 132(13):4678-84 (2010).

31. Buceta M, Domínguez E, Castro M, Brea J, Alvarez D, Barcala

J, Valdés L, Alvarez-Calderón P, Domínguez F, Vidal B, Díaz

JL, Miralpeix M, Beleta J, Cadavid MI, Loza MI. A new chemical tool

(C0036E08) supports the role of adenosine A(2B) receptors in

mediating human mast cell activation. Biochem Pharmacol. 76(7):912-

21 (2008).

32. Bur M, Henning A, Hein S, Schneider M, Lehr CM. Inhalative

nanomedicine--opportunities and challenges. Inhal Toxicol. 1:137-43

(2009).

Referencias

223

33. Calvo P, Alonso MJ, Vila-Jato JL, Robinson JR. Improved Ocular

Bioavailability of Indomethacin by Novel Ocular Drug Carriers. J

Pharm Pharmacol. 48:1147-1152 (1996).

34. Calvo P, Remuñan-López C, Vila-Jato JL, Alonso MJ: Development of

positively charged colloidal drug carriers: Chitosan-coated polyester

nanocapsules and submicron-emulsions. Colloid. Polym. Sci. 275, 46-53

(1997).

35. Calvo P, Remuñan-López C, Vila-Jato JL, Alonso MJ. Chitosan and

chitosan/ethylene oxide-propylene oxide block copolymer nanoparticles

as novel carriers for proteins and vaccines. Pharm Res. 14(10):1431-6

(1997).

36. Calvo P, Remuñan-Lopez C, Vila-Jato JL, Alonso M.J. Novel

hydrophilic chitosan–polyethylene oxide nanoparticles as protein

carriers. J. Appl. Polymer Sci. 63:125–132 (1997).

37. Calvo P, Sanchez A, Martinez J, Lopez MI, Calonge M, Pastor JC,

Alonso MJ. Polyester nanocapsules as new topical ocular delivery

systems for cyclosporin A. Pharm Res. 13: 311-315 (1996).

38. Calvo P, Thomas C, Alonso MJ, Vila-Jato JL, Robinson .R. Study of the

mechanism of interaction of poly(E-caprolactone) nanocapsules with the

cornea by confocal laser scanning microscopy. Int J Pharm. 103:283-

291 (1994).

39. Calvo P, Vila-Jato JL, Alonso MJ. Comparative in vitro evaluation of

several colloidal systems, nanoparticles, nanocapsules, and

nanoemulsions, as ocular drug carriers. J Pharm Sci. 85:530-536 (1996).

40. Calvo P, Vila-Jato JL, Alonso MJ. Evaluation of cationic polymer-

coated nanocapsules as ocular drug carriers. Int J Pharm. 153:41-50

(1997).

Referencias

224

41. Cafaggi S, Russo E, Stefani R, Leardi R, Caviglioli G, Parodi

B, Bignardi G, De Totero D, Aiello C, Viale M. Preparation and

evaluation of nanoparticles made of chitosan or N-trimethyl chitosan

and a cisplatin-alginate complex. J Control Release. 121(1-2):110-23

(2007).

42. Campo C, Molinari JF, Ungo J, Ahmed T. Molecular-weight-dependent

effects of nonanticoagulant heparins on allergic airway responses. J

Appl Physiol. 86(2):549-57 (1999).

43. Cauchetier E, Deniau M, Fessi H, Astier A, Paul M. Atovaquone-loaded

nanocapsules: Influence of the nature of the polymer on their in vitro

characteristics. Int J Pharm. 250: 273-281 (2003).

44. Chen Y, Siddalingappa B, Chan PH, Benson HA.Development of a

chitosan-based nanoparticle formulation for delivery of a hydrophilic

hexapeptide, dalargin. Biopolymers. 90(5):663-70 (2008).

45. Chen MC, Wong HS, Lin KJ, Chen HL, Wey SP, Sonaje K, Lin

YH, Chu CY, Sung HW. The characteristics, biodistribution and

bioavailability of a chitosan-based nanoparticulate system for the oral

delivery of heparin.

46. Choi MJ, Briançon S, Andrieu J, Min SG, Fessi H. Effect of freeze-

drying process conditions on the stability of nanoparticles. Dry.

Technol. 22: 335-346 (2004).

47. Choi KY, Min KH, Na JH et al.: Self-assembled hyaluronic acid

nanoparticles as a potential drug carrier for cancer therapy: synthesis,

characterization, and in vivo biodistribution J. Mater. Chem. 19, 4102-

4107 (2009).

48. Cournarie F, Auchere D, Chevenne D, Lacour B, Seiller M, Vauthier, C.

Absorption and efficiency of insulin after oral administration of insulin-

loaded nanocapsules in diabetic rats. Int J Pharm. 242:325-328 (2002).

Referencias

225

49. Couvreur P, Barratt G, Fattal E, Legrand P, Vauthier C. Nanocapsule

technology: A review. Crit Rev Ther Drug Carrier Syst. 19:99-134

(2002).

50. Couvreur P, Kante B, Roland, M. Polycyanoacrylate nanocapsules as

potential lysosomotropic carriers: Preparation, morphological and

sorptive properties. J Pharm Pharmacol. 31:331-332 (1979).

51. Couvreur P, Tulkens P, Roland, M. Nanocapsules: a new type of

lysosomotropic carrier. FEBS Lett. 84: 323-326 (1977).

52. Cowman MK, Matsuoka S. Experimental approaches to hyaluronan

structure. Carbohydr Res. 340(5):791-809 (2005).

53. Crown J, O'Leary M: The taxanes: An update. Lancet 355(9210), 1176-

8 (2000).

54. Csaba N, Köping-Höggård M, Fernandez-Megia E, Novoa-Carballal

R, Riguera R, Alonso MJ. Ionically crosslinked chitosan nanoparticles

as gene delivery systems: effect of PEGylation degree on in vitro and in

vivo gene transfer. J Biomed Nanotechnol. 5(2):162-71 (2009).

55. Csaba N, Garcia-Fuentes M, Alonso MJ. The performance of

nanocarriers for transmucosal drug delivery. Expert Opin Drug Deliv.

3:463-478 (2006).

56. Csaba N, Garcia-Fuentes M, Alonso MJ. Nanoparticles for nasal

vaccination. Adv Drug Deliv Rev. 61(2):140-57 (2009).

57. Csaba N, Köping-Höggård M, Alonso MJ. Ionically crosslinked

chitosan/tripolyphosphate nanoparticles for oligonucleotide and plasmid

DNA delivery. Int J Pharm. 382(1-2):205-14 (2009).

58. Damge C, Michel C, Aprahamian M, Couvreur P. New approach for

oral administration of insulin with polyalkylcyanoacrylate nanocapsules

as drug carrier. Diabetes. 37:246-251 (1988).

Referencias

226

59. Damge C, Vranckx H, Balschmidt P, Couvreur P. Poly(alkyl

cyanoacrylate) nanospheres for oral administration of insulin. J Pharm

Sci. 86:1403-1409 (1997).

60. De Campos AM, Diebold Y, Carvalho EL, Sanchez A, Alonso MJ.

Chitosan nanoparticles as newocular drug delivery systems: in vitro

stability, in vivo fate, and cellular toxicity. Pharm. Res. 21:803–810

(2004).

61. De Campos AM, Sanchez A, Gref R, Calvo P, Alonso M.J. The effect of

a PEG versus a chitosan coating on the interaction of drug colloidal

carriers with the ocular mucosa. Eur J Pharm Sci. 20:73-81 (2003).

62. De la Fuente M, Csaba N, Garcia-Fuentes M, Alonso MJ. Nanoparticles as protein and gene carriers to mucosal surfaces. Nanomedicine (Lond). 3(6):845-57 (2008).

63. De la Fuente M, Seijo B, Alonso MJ. Novel hyaluronic acid-chitosan

nanoparticles for ocular gene therapy. Invest Ophthalmol Vis Sci.

49(5):2016‐24 (2008).

64. De la Fuente M, Seijo B, Alonso MJ. Bioadhesive hyaluronan–chitosan

nanoparticles can transport genes across the ocular mucosa and transfect

ocular tissue. Gene Ther. 15:668–676 (2008).

65. De la Fuente M, Seijo B, Alonso MJ. Novel hyaluronan-based

nanocarriers for transmucosal delivery of macromolecules. Macromol.

Biosci. 8:441–450 (2008).

66. Densmore CL, Kleinerman ES, Gautam A, Jia SF, Xu B, Worth

LL, Waldrep JC, Fung YK, T'Ang A, Knight V.Growth suppression of

established human osteosarcoma lung metastases in mice by aerosol

gene therapy with PEI-p53 complexes. Cancer Gene Ther. 8(9):619-27

(2001).

67. Des Rieux A, Fievez V, Garinot M, Schneider YJ, Preat V.

Nanoparticles as potential oral delivery systems of proteins and

vaccines: A mechanistic approach. J Control Release. 116:1-27 (2006).

Referencias

227

68. Desai MP, Labhasetwar V, Amidon GL, Levy RJ. Gastrointestinal

Uptake of Biodegradable Microparticles: Effect of Particle Size. Pharm

Res. 13:1838-1845 (1996).

69. Desai SD, Blanchard J. Pluronic® F127-based ocular delivery system

containing biodegradable polyisobutylcyanoacrylate nanocapsules of

pilocarpine. Drug Delivery: J Delivery and Targeting Therapeutic

Agents. 7:201-207 (2000).

70. Dhanikula A, Khalid N, Lee SD, Yeung R, Risovic V, Wasan KM,

Leroux JC. Long circulating lipid nanocapsules for drug detoxification.

Biomaterials. 28:1248-1257 (2007).

71. Diamant Z, Page CP. Heparin and related molecules as a new treatment

for asthma. Pulm Pharmacol Ther. 13(1):1-4 (2000).

72. Di Colo G, Zambito Y, Zaino C, Sansò M.Selected polysaccharides at

comparison for their mucoadhesiveness and effect on precorneal

residence of different drugs in the rabbit model. Drug Dev Ind Pharm.

35(8):941-9 (2009).

73. Edwards DA, Hanes J, Caponetti G, Hrkach J, Ben-Jebria A, Eskew

ML, Mintzes J, Deaver D, Lotan N, Langer R. Large porous particles for

pulmonary drug delivery. Science. 276(5320):1868-71 (1997).

74. Ehdaie B. Application of Nanotechnology in Cancer Research: Review

of Progress in the National Cancer Institute's Alliance for

nanotechnology (2008).

75. Eliaz RE, Nir S, Marty C, Szoka FC Jr. Determination and modeling of

kinetics of cancer cell killing by doxorubicin and doxorubicin

encapsulated in targeted liposomes. Cancer Res. 64:711–718 (2004).

76. Eliaz RE, Nir S, Szoka FC Jr.Interactions of hyaluronan-targeted

liposomes with cultured cells: modeling of binding and endocytosis.

Methods Enzymol. 387:16-33 (2004).

Referencias

228

77. Eliaz RE, Szoka FC Jr. Liposome-encapsulated doxorubicin targeted to

CD44: a strategy to kill CD44-overexpressing tumor cells. Cancer Res.

61(6):2592-601 (2001).

78. Engels FK, Mathot RA, Verweij J. Alternative drug formulations of

docetaxel: a review. Anticancer Drugs. 18(2):95-103 (2007).

79. Fattal E. Bochot A. State of the art and perspectives for the delivery of

antisense oligonucleotides and siRNA by polymeric nanocarriers. Int J

Pharm. 364:237-248 (2008).

80. Fessi H, Puisieux F, Devissaguet JP, Ammoury N, Benita, S.

Nanocapsule formation by interfacial polymer deposition following

solvent deplacement. Int J Pharm. 55:25-28 (1989).

81. Freitas C, Müller RH: Effect of light and temperature on zeta potential

and physical and physical stability in solid lipid nanoparticle (SLNTM)

dispersions. Int. J. Pharm. 168, 221-229 (1998).

82. Fresta M, Cavallaro G, Giammona G, Wehrli E, Puglisi G. Preparation

and characterization of polyethyl-2-cyanoacrylate nanocapsules

containing antiepileptic drugs. Biomaterials. 17:751-758 (1996).

83. Gallardo M, Couarraze G, Denizot B, Treupel L, Couvreur P, Puisieux

F. Study of the mechanisms of formation of nanoparticles and

nanocapsules of polyisobutyl-2-cyanoacrylate. Int J Pharm. 100:55-64

(1993).

84. Garcia-Contreras L, Fiegel J, Telko MJ, Elbert K, Hawi A, Thomas

M, VerBerkmoes J, Germishuizen WA, Fourie PB, Hickey AJ, Edwards

D. Inhaled large porous particles of capreomycin for treatment of

tuberculosis in a guinea pig model. Antimicrob Agents Chemother.

51(8):2830-6 (2007).

85. Garcia-Fuentes M, Prego C, Torres D, Alonso MJ. A comparative study

of the potential of solid triglyceride nanostructures coated with chitosan

or poly(ethylene glycol) as carriers for oral calcitonin delivery. Eur. J.

Pharm. Sci. 25:133–143 (2005).

Referencias

229

86. Garcion E, Lamprecht A, Heurtault B, Paillard A, Aubert-Pouessel

A, Denizot B, Menei P, Benoît JP.A new generation of anticancer, drug-

loaded, colloidal vectors reverses multidrug resistance in glioma and

reduces tumor progression in rats. Mol Cancer Ther. 5(7):1710-22

(2006).

87. Garrigo J, Danta I, Ahmed T. Time course of the protective effect of

inhaled heparin on exercise-induced asthma. Am J Respir Crit Care

Med. 153(5):1702-7 (1996).

88. Gelderblom H, Verweij J, Nooter K, Sparreboom A. Cremophor EL: the

drawbacks and advantages of vehicle selection for drug formulation. Eur

J Cancer. 37(13):1590-8 (2001).

89. Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: Role

of ATP-dependent transporters. Nat Rev Cancer. 2:48-58 (2002).

90. Green WF, Konnaris K, Woolcock AJ. Effect of salbutamol, fenoterol,

and sodium cromoglicate on the release of heparin fromsensitized

human lung fragments challenged with Dermatophagoides pteronyssinus

allergen. Am. J. Respir. Cell Mol. Biol. 8:518–521 (1993).

91. Greish K. Enhanced permeability and retention of macromolecular

drugs in solid tumors: A royal gate for targeted anticancer

nanomedicines. J Drug Target.15:457-464 (2007).

92. Guinebretiere S, Briançon S, Fessi H, Teodorescu VS, Blanchin MG.

Nanocapsules of biodegradable polymers: Preparation and

characterization by direct high resolution electron microscopy. Mater

Sci Eng C. 21:137-142 (2002).

93. Guinebretiere S, Briancon S, Lieto J, Mayer C, Fessi H. Study of the

emulsion-diffusion of solvent: Preparation and characterization of

nanocapsules. Drug Dev Res. 57:18-33 (2002).

94. Halayko AJ, Rector E, Stephens NL. Characterization of molecular

determinants of smooth muscle cell heterogeneity. Can J Physiol

Pharmacol. 75(7):917-29 (1997).

Referencias

230

95. Heinemann L. New ways of insulin delivery. Int J Clin Pract Suppl.

166:29-40 (2010).

96. Heurtault B, Saulnier P, Pech B, Benoit JP, Proust JE. Interfacial

stability of lipid nanocapsules. Colloids Surf B Biointerfaces. 30:225-

235 (2003).

97. Heurtault B, Saulnier P, Pech B, Proust JE, Benoit, JP. A novel phase

inversion-based process for the preparation of lipid nanocarriers. Pharm

Res. 19:875-880 (2002).

98. Heurtault B, Saulnier P, Pech B, Proust JE, Benoit JP: Properties of

polyethylene glycol 660 12-hydroxy stearate at a triglyceride/water

interface. Int. J. Pharm. 242(1-2), 167-70 (2002).

99. Heurtault B.; Saulnier P.; Pech B.; Proust J.E.; Benoit J.P. Physico-

chemical stability of colloidal lipid particles. Biomaterials. 24:4283-

4300 (2003).

100. Heurtault B, Saulnier P, Pech B, Venier-Julienne MC, Proust JE, Phan-

Tan-Luu R, Benoit JP. The influence of lipid nanocapsule composition

on their size distribution. Eur J Pharm Sci. 18:55-61 (2003).

101. Hiemenz PC, Rajagopalan R. Principles of Colloidal and Surface

Chemistry, 3rd edition (1997). Dekker, New York, USA (Libro).

102. Hillaireau H, Le Doan T, Chacun H, Janin J, Couvreur P. Encapsulation

of mono- and oligo-nucleotides into aqueous-core nanocapsules in

presence of various water-soluble polymers. Int J Pharm. 331:148-152

(2007).

103. Hitzman CJ, Wattenberg LW, Wiedmann TS. Pharmacokinetics of 5-

fluorouracil in the hamster following inhalation delivery of lipid-coated

nanoparticles. J Pharm Sci. 95(6):1196-211 (2006).

104. Hohenegger M. Novel and current treatment concepts using pulmonary

drug delivery. Curr Pharm Des. 16(22):2484-92 (2010).

Referencias

231

105. Hong GY, Jeong YI, Lee SJ, Lee E, Oh JS, Lee HC. Combination of

paclitaxel- and retinoic acid-incorporated nanoparticles for the treatment

of CT-26 colon carcinoma. Arch Pharm Res. 34(3):407-17 (2011).

106. Hureaux J, Lagarce F, Gagnadoux F, Vecellio L, Clavreul A, Roger E,

Kempf M, Racineux JL, Diot P, Benoit JP, Urban T. Lipid

nanocapsules: ready-to-use nanovectors for the aerosol delivery of

paclitaxel. Eur J Pharm Biopharm. 73(2):239-46 (2009).

107. Hureaux J, Lagarce F, Gagnadoux F, Rousselet MC, Moal

V, Urban T, Benoit JP. Toxicological study and efficacy of blank

and paclitaxel-loaded lipid nanocapsules after i.v. administration

in mice. Pharm Res. 27(3):421-30 (2010).

108. Huynh NT, Passirani C, Saulnier P, Benoit JP. Lipid nanocapsules: a

new platform for nanomedicine. Int J Pharm. 379(2):201-9 (2009).

109. Hyung W, Ko H, Park J, Lim E, Park SB, Park YJ, Yoon HG, Suh

JS, Haam S, Huh YM. Novel hyaluronic acid (HA) coated drug carriers

(HCDCs) for human breast cancer treatment. Biotechnol Bioeng.

99(2):442-54 (2008).

110. Immordino ML, Brusa P, Arpicco S, Stella B, Dosio F, Cattel

L.Preparation, characterization, cytotoxicity and pharmacokinetics of

liposomes containing docetaxel. J Control Release. 91(3):417-29 (2003).

111. Issa MM, Köping-Höggård M, Tømmeraas K, Vårum KM, Christensen

BE, Strand SP, Artursson P. Targeted gene delivery with trisaccharide-

substituted chitosan oligomers in vitro and after lung administration in

vivo. J Control Release. 115(1):103-12 (2006).

112. Iyer AK, Khaled G, Fang J, Maeda H. Exploiting the enhanced

permeability and retention effect for tumor targeting. Drug Discov

Today. 11:812-818 (2006).

113. Jaques LB. Heparins--anionic polyelectrolyte drugs. Pharmacol Rev.

31(2):99-166 (1979).

Referencias

232

114. Jin H, Kim TH, Hwang SK, Chang SH, Kim HW, Anderson HK, Lee

HW, Lee KH, Colburn NH, Yang HS, Cho MH, Cho CS. Aerosol

delivery of urocanic acid-modified chitosan/programmed cell death 4

complex regulated apoptosis, cell cycle, and angiogenesis in lungs of K-

ras null mice. Mol Cancer Ther. 5(4):1041-9 (2006).

115. Johnson PR, Armour CL, Carey D, Black JL. Heparin and PGE2 inhibit

DNA synthesis in human airway smooth muscle cells in culture. Am J

Physiol. 269(4 Pt 1):L514-9 (1995).

116. Jones SE, Erban J, Overmoyer B, Budd GT, Hutchins L, Lower

E, Laufman L, Sundaram S, Urba WJ, Pritchard KI, Mennel R, Richards

D, Olsen S, Meyers ML, Ravdin PM. Randomized phase III study of

docetaxel compared with paclitaxel in metastatic breast cancer. J Clin

Oncol. 23(24):5542-51 (2005).

117. Kanabar V, Hirst SJ, O'Connor BJ, Page CP. Some structural

determinants of the antiproliferative effect of heparin-like molecules on

human airway smooth muscle. Br J Pharmacol. 146(3):370-7 (2005).

118. Khalid MN, Simard P, Hoarau D, Dragomir A, Leroux JC. Long

circulating poly(ethylene glycol)-decorated lipid nanocapsules deliver

docetaxel to solid tumors. Pharm Res. 23(4):752-8 (2006).

119. Kilfeather SA, Tagoe S, Perez AC, Okona-Mensa K, Matin R, Page CP.

Inhibition of serum-induced proliferation of bovine tracheal smooth

muscle cells in culture by heparin and related glycosaminoglycans. Br J

Pharmacol. 114(7):1442-6 (1995).

120. Kimura S, Egashira K, Chen L, Nakano K, Iwata E, Miyagawa

M, Tsujimoto H, Hara K, Morishita R, Sueishi K, Tominaga

R, Sunagawa K. Nanoparticle-mediated delivery of nuclear factor

kappaB decoy into lungs ameliorates monocrotaline-induced pulmonary

arterial hypertension. Hypertension. 53(5):877-83 (2009).

Referencias

233

121. Köping-Höggård M, Issa MM, Köhler T, Tronde A, Vårum

KM, Artursson P. A miniaturized nebulization catheter for improved

gene delivery to the mouse lung. J. Gene Med. 7(9):1215-22 (2005).

122. Köping-Höggård M, Sanchez A, Alonso MJ. Nanoparticles as carriers

for nasal vaccine delivery. Exp. Rev. Vaccines 4:185–196 (2005).

123. Krauland AH, Alonso MJ. Chitosan/cyclodextrin nanoparticles as

macromolecular drug delivery system. Int J Pharm. 340(1-2):134-42

(2007).

124. Kumar MN, Mohapatra SS, Kong X, Jena PK, Bakowsky U, Lehr CM.

Cationic poly(lactide-co-glycolide) nanoparticles as efficient in vivo

gene transfection agents. J Nanosci Nanotechnol. 4(8):990-4 (2004).

125. Kurmi BD, Kayat J, Gajbhiye V, Tekade RK, Jain NK. Micro- and

nanocarrier-mediated lung targeting. Expert Opin Drug Deliv. 7(7):781-

94 (2010).

126. Lacoeuille F, Hindre F, Moal F, Roux J, Passirani C, Couturier O, Cales

P, Le Jeune JJ, Lamprecht A, Benoit JP. In vivo evaluation of lipid

nanocapsules as a promising colloidal carrier for paclitaxel. Int J

Pharm. 344(1-2):143-9 (2007).

128. Lago J, Alfonso A, Vieytes MR, Botana LM. Ouabain-induced

enhancement of rat mast cells response. Modulation by protein

phosphorylation and intracellular pH. Cell Signal. 13(7):515-24 (2001).

129. Lambert G, Bertrand JR, Fattal E, Subra F, Pinto-Alphandary H, Malvy

C, Auclair C, Couvreur P. EWS Fli-1 antisense nanocapsules inhibits

Ewing sarcoma-related tumor in mice. Biochem Biophys Res

Commun.279:401-406 (2000).

130. Lambert G, Fattal E, Pinto-Alphandary H, Gulik A, Couvreur P.

Polyisobutylcyanoacrylate nanocapsules containing an aqueous core as a

novel colloidal carrier for the delivery of oligonucleotides. Pharm Res.

17:707-714 (2000).

Referencias

234

131. Lambert G, Fattal E, Pinto-Alphandary H, Gulik A, Couvreur P.

Polyisobutylcyanoacrylate nanocapsules containing an aqueous core for

the delivery of oligonucleotides. Int J Pharm. 214:13-16 (2001).

132. Lang JC. Ocular drug delivery conventional ocular formulations. Adv

Drug Deliv Rev. 16:39-43 (1995).

133. Le Bourlais CA, Chevanne F, Turlin B, Acar L, Zia H, Sado PA,

Needham TE, Leverge R. Effect of cyclosporine A formulations on

bovine corneal absorption: Ex-vivo study. J Microencapsulation.

14:457-467 (1997).

134. Lee DW, Yun KS, Ban HS, Choe W, Lee SK, Lee KY. Preparation and

characterization of chitosan/polyguluronate nanoparticles for siRNA

delivery. J Control Release. 139(2):146-52 (2009).

136. Lee SH, Yoo SD, Lee KH: Rapid and sensitive determination of

paclitaxel in mouse plasma by high-performance liquid chromatography.

J. Chromatogr., B: Biomed. Sci. Appl., 724, 357-363 (1999).

137. Legrand P, Barratt G, Mosqueira V, Fessi H, Devissaguet, JP. Polymeric

nanocapsules as drug delivery systems: A review. STP Pharm. Sci.

9:411-418 (1999).

138. Lenaerts V, Labib A, Chouinard F, Rousseau J, Ali H, Van Lier J.

Nanocapsules with a reduced liver uptake: Targeting of phthalocyanines

to EMT-6 mouse mammary tumor in vivo. Eur J Pharm Biopharm.

41:38-43 (1995).

139. Li X. Chan WK. Transport, metabolism and elimination mechanisms of

anti-HIV agents. Adv Drug Deliv Rev. 39:81-103 (1999).

140. Liang G, Jia-Bi Z, Fei X, Bin N. Preparation, characterization and

pharmacokinetics of N-palmitoyl chitosan anchored docetaxel

liposomes. J Pharm Pharmacol. 59(5):661-7 (2007).

141. Lim ST, Martin GP, Berry DJ, Brown MB. Preparation and evaluation

of the in vitro drug release properties and mucoadhesion of novel

Referencias

235

microspheres of hyaluronic acid and chitosan. J Control Release. 66(2-

3):281-92 (2000).

142. Limayem Blouza I, Charcosset C, Sfar S, Fessi H. Preparation and

characterization of spironolactone-loaded nanocapsules for paediatric

use. Int J Pharm. 325:124‐131 (2006).

143. Lindahl U, Bäckström G, Thunberg L. The antithrombin-binding

sequence in heparin. Identification of an essential 6-O-sulfate

group. J Biol Chem. 258(16):9826-30 (1983).

143. Liu D, Wang L, Liu Z, Zhang C, Zhang N. Preparation, characterization,

and in vitro evaluation of docetaxel-loaded poly(lactic acid)-

poly(ethylene glycol) nanoparticles for parenteral drug delivery. J

Biomed Nanotechnol. 6(6):675-82 (2010).

144. Lopez-Leon T, Carvalho EL, Seijo B, Ortega-Vinuesa JL, Bastos-

Gonzalez D. Physicochemical characterization of chitosan

nanoparticles: electrokinetic and stability behavior. J. Colloid Interface

Sci. 283:344–351 (2005).

144. Losa C, Marchal-Heussler L, Orallo F, Vila-Jato JL, Alonso MJ.

Design of new formulations for topical ocular administration:

Polymeric nanocapsules containing metipranolol. Pharm Res.

10:80-87 (1993).

145. Lowe PJ, Temple CS. Calcitonin and insulin in isobutylcyanoacrylate

nanocapsules: Protection against proteases and effect on intestinal

absorption in rats. J Pharm Pharmacol. 46:547-552 (1994).

146. Lozano MV, Torrecilla D, Lallana E, Vidal A, Fernández-Megía R,

Riguera R, Dominguez F, Alonso M J and Torres D. Chitosan

nanocapsules for active tumor targeting, 7th World Meeting on

Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology,

Malta, 2010.

Referencias

236

147. Lozano MV, Torrecilla D, Torres D, Vidal A, Domínguez F, Alonso

MJ. Highly efficient system to deliver taxanes into tumor cells:

docetaxel-loaded chitosan oligomer colloidal carriers.

Biomacromolecules. 9(8):2186-93 (2008).

148. Lozano MV, Lollo G, Brea J, Torres D, Loza MI, Alonso MJ. Freeze-

dried polysaccharide nanocapsules: efficient vehicles for the

intracellular delivery of docetaxel. (submitted, 2011).

149. Lozano MV, Lollo G, Brea J, Torres D, Loza MI, Alonso MJ.

Polyarginine nanocapsules: a new platform for intracellular drug

delivery (submitted, 2011).

150. Lucio J, D'Brot J, Guo CB, Abraham WM, Lichtenstein

LM, Kagey-Sobotka A, Ahmed T. Immunologic mast cell-

mediated responses and histamine release are attenuated by

heparin. J Appl Physiol. 73(3):1093-101 (1992).

151. Luo Y, Bernshaw NJ, Lu ZR, Kopecek J, Prestwich GD. Targeted

delivery of doxorubicin by HPMA copolymer-hyaluronan

bioconjugates. Pharm Res. 19(4):396-402 (2002).

152. Luo Y, Prestwich GD. Synthesis and selective cytotoxicity of a

hyaluronic acid-antitumor bioconjugate. Bioconjug. Chem. 10, 755–763

(1999).

153. Ma Z, Lim LY. Uptake of chitosan and associated insulin in Caco-2 cell

monolayers: a comparison between chitosan molecules and chitosan

nanoparticles. Pharm Res. 20(11):1812-9 (2003).

154. Mansour HM, Rhee YS, Wu X. Nanomedicine in pulmonary delivery.

Int J Nanomedicine. 4:299-319 (2009).

155. Marchal-Heussler L, Fessi H, Devissaguet JP, Hoffman M, Maincent P.

Colloidal drug delivery systems for the eye. A comparison of the

efficacy of three different polymers: Polyisobutylcyanoacrylate,

Referencias

237

polylactic-co-glycolic acid, poly-epsilon-caprolactone. STP Pharma

Sciences. 2:98-104 (1992).

156. Marchal-Heussler L, Sirbat D, Hoffman M, Maincent P. Poly(ε-

caprolactone) nanocapsules in carteolol ophthalmic delivery. Pharm

Res. 10:386-390 (1993).

157. Martinez-Salas J, Mendelssohn R, Abraham WM, Hsiao B, Ahmed T.

Inhibition of allergic airway responses by inhaled low-molecular-weight

heparins: molecular-weight dependence. J Appl Physiol. 84(1):222-8

(1998).

158. McNeil SE. Nanotechnology for the biologist. J Leukoc Biol. 78:585-

594 (2005).

159. Misra A, Hickey AJ, Rossi C, Borchard G, Terada H, Makino K, Fourie

PB, Colombo P. Inhaled drug therapy for treatment of tuberculosis.

Tuberculosis (Edinb). 91(1):71-81 (2011).

160. Mitchison DA, Fourie PB. The near future: improving the activity of

rifamycins and pyrazinamide. Tuberculosis (Edinb). 90(3):177-81

(2010).

161. Moinard-Checot D, Chevalier Y, Briancon S, Beney L, Fessi H.

Mechanism of nanocapsules formation by the emulsion-diffusion

process. J Colloid Interface Sci. 317:458-468 (2008).

162. Molinari JF, Campo C, Shakir S, Ahmed T. Inhibition of antigen-

induced airway hyperresponsiveness by ultralow molecular-weight

heparin. Am J Respir Crit Care Med. 157(3 Pt 1):887-93 (1998).

163. Mora-Huertas CE, Fessi H, Elaissari A. Polymer-based nanocapsules for

drug delivery. Int J Pharm. 385(1-2):113-42 (2010).

164. Mosqueira VCF, Legrand P, Gulik A, Bourdon O, Gref R, Labarre D,

Barratt G. Relationship between complement activation, cellular uptake

and surface physicochemical aspects of novel PEG-modified

nanocapsules. Biomaterials. 22:2967-2979 (2001).

Referencias

238

165. Mosqueira VCF, Legrand P, Morgat JL, Vert M, Mysiakine E, Gref R,

Devissaguet JP, Barratt G. Biodistribution of long-circulating PEG-

grafted nanocapsules in mice: Effects of PEG chain length and density.

Pharm Res. 18:1411-1419 (2001).

166. Mossman T: Rapid colorimetric assay for cellular growth and survival:

application to proliferation and cytotoxicity assays. J. Immunol.

Methods 65, 55-63 (1983).

167. Motlekar NA, Youan BB. The quest for non-invasive delivery of

bioactive macromolecules: a focus on heparins. J Control Release.

113(2):91-101 (2006).

168. Nafee N, Taetz S, Schneider M, Schaefer UF, Lehr CM. Chitosan-

coated PLGA nanoparticles for DNA/RNA delivery: effect of the

formulation parameters on complexation and transfection of antisense

oligonucleotides. Nanomedicine. 3(3):173-83 (2007).

169. Nakada Y, Fattal E, Foulquier M, Couvreur P. Pharmacokinetics and

biodistribution of oligonucleotide adsorbed onto

poly(isobutylcyanoacrylate) nanoparticles after intravenous

administration in mice. Pharm Res. 13:38-43 (1996).

170. Naor D, Nedvetzki S, Golan I, Melnik L, Faitelson Y. CD44 in cancer.

Crit Rev Clin Lab Sci. 39(6):527-79 (2002).

171. Nassar T, Attili-Qadri S, Harush-Frenkel O, Farber S, Lecht

S, Lazarovici P, Benita S. High plasma levels and effective lymphatic

uptake of docetaxel in an orally available nanotransporter formulation.

Cancer Res. 71(8):3018-28 (2011).

172. Nassar T, Rom A, Nyska A, Benita S. A novel nanocapsule delivery

system to overcome intestinal degradation and drug transport limited

absorption of P-glycoprotein substrate drugs. Pharm Res. 25:2019-2029

(2008).

Referencias

239

173. Nassar T, Rom A, Nyska A, Benita S. Novel double coated

nanocapsules for intestinal delivery and enhanced oral bioavailability of

tacrolimus, a P-gp substrate drug. J Control Release. 133:77-84 (2009).

174. Neumiller JJ, Campbell RK.Technosphere insulin: an inhaled prandial

insulin product. BioDrugs. 2010 Jun;24(3):165-72. doi:

10.2165/11536700-000000000-00000.

175. Niven AS, Argyros G. Alternate treatments in asthma. Chest.

123(4):1254-65 (2003).

176. Ohashi K, Kabasawa T, Ozeki T, Okada H. One-step preparation of

rifampicin/poly(lactic-co-glycolic acid) nanoparticle-containing

mannitol microspheres using a four-fluid nozzle spray drier for

inhalation therapy of tuberculosis. J Control Release. 135(1):19-24.

(2009).

177. Ortner MJ, Chignell CF. The effect of concentration on the binding of

compound 48/80 to rat mast cells: a fluorescence microscopy study.

Immunopharmacology. 3(3):187-91 (1981).

178. Ossipov DA: Nanostructured hyaluronic acid-based materials for active

delivery to cancer. Expert Opin. Drug Deliv. 7(6), 681-703 (2010).

179. Oyarzun-Ampuero FA, Brea J, Loza MI, Torres D, Alonso MJ.


treatment of asthma. Int J Pharm. 381(2):122-9 (2009).

180. Page CP. One explanation of the asthma paradox: inhibition of natural

anti-inflammatory mechanism by beta 2-agonist. Lancet. 337:717–720

(1991).

181. Page S, Ammit AJ, Black JL, Armour CL. Human mast cell and airway

smooth muscle cell interactions: implications for asthma. Am. J.

Physiol. Lung Cell. Mol. Physiol. 281: L1313–L1323 (2001).

182. Pandey R, Sharma A, Zahoor A, Sharma S, Khuller GK, Prasad B. Poly

(DL-lactide-co-glycolide) nanoparticle-based inhalable sustained drug

Referencias

240

delivery system for experimental tuberculosis. J Antimicrob Chemother.

52(6):981-6 (2003).

183. Pandita D, Ahuja A, Lather V, Dutta T, Velpandian T, Khar RK.

Development, characterization and in vitro assessement of stearylamine-

based lipid nanoparticles of paclitaxel. Pharmazie. 66(3):171-7 (2011).

184. Paraskar AS, Soni S, Chin KT, Chaudhuri P, Muto KW, Berkowitz

J, Handlogten MW, Alves NJ, Bilgicer B, Dinulescu DM, Mashelkar

RA, Sengupta S. Harnessing structure-activity relationship to engineer a

cisplatin nanoparticle for enhanced antitumor efficacy. Proc Natl Acad

Sci U S A. 107(28):12435-40 (2010).

185. Peer D, Margalit R.Tumor-targeted hyaluronan nanoliposomes increase

the antitumor activity of liposomal Doxorubicin in syngeneic and human

xenograft mouse tumor models. Neoplasia. 6(4):343-53 (2004).

186. Peer D, Margalit R: Loading mitomycin C inside long circulating

hyaluronan targeted nano-liposomes increases its antitumor activity in

three mice tumor models. Int. J. Cancer 108:780-789 (2004).

187. Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R:

Nanocarriers as an emerging platform for cancer therapy. Nat.

Nanotechnol. 2(12), 751-60 (2007).

188. Peltier S, Oger JM, Lagarce F, Couet W, Benoît JP.Enhanced oral

paclitaxel bioavailability after administration of paclitaxel-loaded lipid

nanocapsules. Pharm Res. 23(6):1243-50 (2006).

189. Penno MB, August JT, Baylin SB, Mabry M, Linnoila RI, Lee

VS, Croteau D, Yang XL, Rosada C. Expression of CD44 in human

lung tumors. Cancer Res. 54(5):1381-7 (1994).

190. Pinto-Alphandary H, Aboubakar M, Jaillard D, Couvreur P, Vauthier C.

Visualization of insulin-loaded nanocapsules: In vitro and in vivo

studies after oral administration to rats. Pharm Res. 20:1071-1084

(2003).

Referencias

241

191. Pinto Reis C, Neufeld RJ, Ribeiro AJ, Veiga F. Nanoencapsulation II.

Biomedical applications and current status of peptide and protein

nanoparticulate delivery systems. Nanomedicine. 2:53-65 (2006).

192. Prego C, Fabre M, Torres D, Alonso M.J. Efficacy and mechanism of

action of chitosan nanocapsules for oral peptide delivery. Pharm Res

.23:549-556 (2006).

193. Prego C, Fernandez-Megia E, Novoa-Carballal R, Quinoa E, Torres D,

Alonso MJ. Chitosan and chitosan-PEG nanocapsules: new carriers for

improving the oral absorption of calcitonin. Presented at: 30th Annual

Meeting of the Controlled Release Society, Glasgow, UK, 19-23 July

2003.

194. Prego C, García M, Torres D, Alonso MJ. Transmucosal

macromolecular drug delivery. J Control Release. 101(1-3):151-62

(2005).

195. Prego C, Torres D, Alonso M.J. Chitosan nanocapsules: A new carrier

for nasal peptide delivery. JDDST. 16:331-337 (2006).

196. Prego C, Torres D, Alonso MJ. Chitosan nanocapsules as carriers for

oral peptide delivery: Effect of chitosan molecular weight and type of

salt on the in vitro behaviour and in vivo effectiveness. J Nanosci

Nanotechnol. 6:2921-2928 (2006).

197. Prego C, Torres D, Fernandez-Megia E, Novoa-Carballal R, Quiñoá

E, Alonso MJ. Chitosan-PEG nanocapsules as new carriers for oral

peptide delivery. Effect of chitosan pegylation degree. J Control

Release. 111(3):299-308 (2006).

198. Preetz C, Rube A, Reiche I, Hause G, Mader K. Preparation and

characterization of biocompatible oil-loaded polyelectrolyte

nanocapsules. Nanomedicine. 4:106-114 (2008).

199. Pritchard, K., Lansley, A.B., Martin, G.P., Helliwell, M., Marriott, C.,

Benedetti, L.M. Evaluation of the bioadhesive properties of hyaluronan

Referencias

242

derivatives:detachment weight and mucociliary transport rate studies.

Int. J. Pharm. 129,137–145 (1996).

200. Puglisi G, Fresta HTM, Giammona G, Ventura CA. Influence of the

preparation conditions on poly(ethylcyanoacrylate) nanocapsule

formation. Int J Pharm. 125:283-287 (1995).

201. Qin Y, Song QG, Zhang ZR, Liu J, Fu Y, He Q, Liu J. Ovarian tumor

targeting of docetaxel-loaded liposomes mediated by luteinizing

hormone-releasing hormone analogues. In vive distribution in nude

mice. Arzneimittelforschung. 58(10):529-34 (2008).

202. Quintanar-Guerrero D, Allemann E, Doelker E, Fessi, H. Preparation

and characterization of nanocapsnles from preformed polymers by a

new process based on emulsification-diffusion technique. Pharm Res.

15:1056-1062 (1998).

203. Raviña M, Cubillo E, Olmeda D, Novoa-Carballal R, Fernandez-Megia

E, Riguera R, Sánchez A, Cano A, Alonso MJ. Hyaluronic

acid/chitosan-g-poly(ethylene glycol) nanoparticles for gene therapy: an

application for pDNA and siRNA delivery. Pharm Res. 27(12):2544-55

(2010).

204. Robinson DS. The role of the mast cell in asthma: induction of airway

hyperresponsiveness by interaction with smooth muscle? J. Allergy

Clin. Immunol. 114:58–65 (2004).

205. Rosato A, Banzato A, De Luca G, Renier D, Bettella F, Pagano

C, Esposito G, Zanovello P, Bassi P. HYTAD1-p20: a new paclitaxel-

hyaluronic acid hydrosoluble bioconjugate for treatment of superficial

bladder cancer. 65 Urol Oncol. 24(3):207-15 (2006).

206. Rowe RC, Sheskey PJ, Quinn. Handbook of pharmaceutical excipients,

6th edition (2009). pharmaceutical press, United Kingdom (libro).

207. Rowinsky EK, Donehower RC. Paclitaxel (taxol). N Engl J Med.

332(15):1004-14 (1995).

Referencias

243

207. Rube A, Hause G, Mader K, Kohlbrecher J. Core-shell structure of

Miglyol/poly(D,L-lactide)/Poloxamer nanocapsules studied by small-

angle neutron scattering. J Control Release. 107:244-252 (2005).

209. Rusch V, Klimstra D, Venkatraman E, Pisters PW, Langenfeld

J, Dmitrovsky E. Overexpression of the epidermal growth factor

receptor and its ligand transforming growth factor alpha is frequent in

resectable non-small cell lung cancer but does not predict tumor

progression. Clin Cancer Res. 3(4):515-22 (1997).

210. Santander-Ortega MJ, Peula-García JM, Goycoolea FM, Ortega-

Vinuesa JL: Chitosan nanocapsules: Effect of chitosan molecular weight

and acetylation degree on electrokinetic behaviour and colloidal

stability. Colloids Surf. B Biointerfaces. 82(2), 571-80 (2011).

211. Saso L, Bonanni G, Grippa E, Gatto MT, Leone MG, Silvestrini

B.Interaction of hyaluronic acid with mucin, evaluated by gel

permeation chromatography. Res Commun Mol Pathol Pharmacol.

104(3):277-84 (1999).

212. Schwab G, Chavany C, Duroux I, Goubin G, Lebeau J, Helene C,

Saison-Behmoaras T. Antisense oligonucleotides adsorbed to

polyalkylcyanoacrylate nanoparticles specifically inhibit mutated Ha-

ras-mediated cell proliferation and tumorigenicity in nude mice. Proc

Natl Acad Sci U S A. 91:10460-10464 (1994).

213. Sharma A, Sharma S, Khuller GK. Lectin-functionalized poly (lactide-

co-glycolide) nanoparticles as oral/aerosolized antitubercular drug

carriers for treatment of tuberculosis. J Antimicrob Chemother.

54(4):761-6 (2004).

214. Sparreboom A, Scripture CD, Trieu V, Williams PJ, De T, Yang

A, Beals B, Figg WD, Hawkins M, Desai N. Comparative preclinical

and clinical pharmacokinetics of a cremophor-free, nanoparticle

albumin-bound paclitaxel (ABI-007) and paclitaxel formulated in

Cremophor (Taxol). Clin Cancer Res. 11(11):4136-43 (2005).

Referencias

244

215. Staniscuaski Guterres S, Fessi H, Barratt G, Puisieux F, Devissaguet JP.

Poly(D,L-Lactide) nanocapsules containing non-steroidal anti-

inflammatory drugs: Gastrointestinal tolerance following intravenous

and oral administration. Pharm Res. 12:1545-1547 (1995).

216. Surace C, Arpicco S, Dufaÿ-Wojcicki A, Marsaud V, Bouclier C, Clay

D, Cattel L, Renoir JM, Fattal E. Lipoplexes targeting the CD44

hyaluronic acid receptor for efficient transfection of breast cancer cells.

Mol Pharm. 6(4):1062-73 (2009).

217. Taetz S, Bochot A, Surace C, Arpicco S, Renoir JM, Schaefer UF,

Marsaud V, Kerdine-Roemer S, Lehr CM, Fattal E. Hyaluronic acid-

modified DOTAP/DOPE liposomes for the targeted delivery of anti-

telomerase siRNA to CD44-expressing lung cancer cells.

Oligonucleotides. 19(2):103-16 (2009).

218.Taetz S, Nafee N, Beisner J, Piotrowska K, Baldes C, MürdterTE, Huwer

H, Schneider M, Schaefer UF, Klotz U, Lehr CM.The influence of

chitosan content in cationic chitosan/PLGA nanoparticles on the

delivery efficiency of antisense 2'-O-methyl-RNA directed against

telomerase in lung cancer cells. Eur J Pharm Biopharm. 72(2):358-69

(2009).

219. Teijeiro-Osorio D, Remuñán-López C, Alonso MJ.

Chitosan/cyclodextrin nanoparticles can efficiently transfect the

airway epithelium in vitro. Eur J Pharm Biopharm. 71(2):257-63

(2009).

220. Teijeiro-Osorio D, Remuñán-López C, Alonso MJ.New generation of

hybrid poly/oligosaccharide nanoparticles as carriers for the nasal

delivery of macromolecules. Biomacromolecules. 10(2):243-9 (2009).

221. Ten Tije AJ, Verweij J, Loos WJ, Sparreboom A: Pharmacological

effects of formulation vehicles : implications for cancer chemotherapy.

Clin. Pharmacokinet. 42(7), 665-85 (2003).

Referencias

245

222. Tomoda K, Ohkoshi T, Hirota K, Sonavane GS, Nakajima T, Terada

H, Komuro M, Kitazato K, Makino K.Preparation and properties of

inhalable nanocomposite particles for treatment of lung cancer. Colloids

Surf B Biointerfaces. 71(2):177-82 (2009).

223. Torrecilla D, Lozano MV, Lallana E, Novoa-Carballal R, Vidal A,

Torres D, Fernández-Megía E, Riguera E, Alonso MJ, Domínguez F

(Sometido, 2011).

224. Toub N, Bertrand JR, Malvy C, Fattal E, Couvreur P. Antisense

oligonucleotide nanocapsules efficiently inhibit EWS-Fli1 expression in

a Ewing's sarcoma model. Oligonucleotides. 16:158-168 (2006).

225. Toub N, Bertrand JR, Tamaddon A, Elhamess H, Hillaireau H,

Maksimenko A, Maccario J, Malvy C, Fattal E, Couvreur P. Efficacy of

siRNA nanocapsules targeted against the EWS-Fli1 oncogene in Ewing

sarcoma. Pharm Res. 23:892-900 (2006).

226. Tran TA, Kallakury BV, Sheehan CE, Ross JS. Expression of CD44

standard form and variant isoforms in non-small cell lung carcinomas.

Hum Pathol. 28(7):809-14 (1997).

227. Trudeau ME, Eisenhauer EA, Higgins BP, Letendre F, Lofters

WS, Norris BD, Vandenberg TA, Delorme F, Muldal AM. Docetaxel in

patients with metastatic breast cancer: a phase II study of the National

Cancer Institute of Canada-Clinical Trials Group. J Clin Oncol.

14(2):422-8 (1996).

228. Tseng CL, Su WY, Yen KC, Yang KC, Lin FH. The use of biotinylated-

EGF-modified gelatin nanoparticle carrier to enhance cisplatin

accumulation in cancerous lungs via inhalation. Biomaterials.

30(20):3476-85 (2009).

229. Tyrell DJ, Kilfeather S, Page CP. Therapeutic uses of heparin beyond its

traditional role as an anticoagulant. Trends Pharmacol Sci. 16(6):198-

204 (1995).

Referencias

246

230. Ubrich N, Schmidt C, Bodmeier R, Hoffman M, Maincent P. Oral

evaluation in rabbits of cyclosporin-loaded Eudragit RS or RL

nanoparticles. Int J Pharm. 288:169-175 (2005).

231. van Zuylen L, Verweij J, Sparreboom A. Role of formulation vehicles in

taxane pharmacology. Invest New Drugs. 19(2):125-41 (2001).

232. Vauthier C, Bouchemal K. Methods for the preparation and manufacture

of polymeric nanoparticles. Pharm Res. 26(5):1025-58 (2009).

233. Vauthier C, Labarre D, Ponchel G. Design aspects of

poly(alkylcyanoacrylate) nanoparticles for drug delivery. J Drug Target.

15:641-663 (2007).

234. Vila A, Gill H, McCallion O, Alonso MJ. Transport of PLA-PEG

particles across the nasal mucosa: effect of particle size and PEG

coating density. J Control Release. 98:231-244 (2004).

235. Vila A, Sánchez A, Janes K, Behrens I, Kissel T, Vila Jato JL, Alonso

MJ. Low molecular weight chitosan nanoparticles as new carriers for

nasal vaccine delivery in mice. Eur J Pharm Biopharm. 57(1):123-31

(2004).

236. Vranckx H, Demoustier M, Deleers M. A new nanocapsule formulation

with hydrophilic core: Application to the oral administration of salmon

calcitonin in rats. Eur J Pharm Biopharm. 42:345-347 (1996).

237. Watnasirichaikul S, Davies NM, Rades T, Tucker IG. Preparation of

biodegradable insulin nanocapsules from biocompatible

microemulsions. Pharm Res. 17:684-689 (2000).

238. Wohlgemuth M, Mayer C. Pulsed field gradient NMR on

polybutylcyanoacrylate nanocapsules. J Colloid Interface Sci. 260:324-

331 (2003).

239. Wong WS, Koh DS. Advances in immunopharmacology of asthma.

Biochem Pharmacol. 59(11):1323-35 (2000).

240. Xin D, Wang Y, Xiang J. The use of amino acid linkers in the

conjugation of paclitaxel with hyaluronic acid as drug delivery system:

Referencias

247

synthesis, self-assembled property, drug release, and in vitro efficiency.

Pharm Res. 27(2):380-9 (2010).

241. Xu CX, Jere D, Jin H, Chang SH, Chung YS, Shin JY, Kim JE, Park

SJ, Lee YH, Chae CH, Lee KH, Beck GR Jr, Cho CS, Cho MH.

Poly(ester amine)-mediated, aerosol-delivered Akt1 small interfering

RNA suppresses lung tumorigenesis. Am J Respir Crit Care Med.

178(1):60-73 (2008).

242. Yamamoto H, Kuno Y, Sugimoto S, Takeuchi H, Kawashima Y.

Surface-modified PLGA nanosphere with chitosan improved pulmonary

delivery of calcitonin by mucoadhesion and opening of the intercellular

tight junctions. J Control Release. 102(2):373-81 (2005).

243. Yamamoto Y, Yoshida M, Sato M, Sato K, Kikuchi S, Sugishita

H, Kuwabara J, Matsuno Y, Kojima Y, Morimoto M, Horiuchi

A, Watanabe Y. Feasibility of tailored, selective and effective anticancer

chemotherapy by direct injection of docetaxel-loaded immunoliposomes

into Her2/neu positive gastric tumor xenografts. Int J Oncol. 38(1):33-9

(2011).

244. Yi D, Wiedmann TS. Inhalation adjuvant therapy for lung cancer. J

Aerosol Med Pulm Drug Deliv. 23(4):181-7 (2010).

245. Yin Win K, Feng S. Effects of particle size and surface coating on

cellular uptake of polymeric nanoparticles for oral delivery of anticancer

drugs. Biomaterials. 26:2713-2722 (2005).

246. Yuan Z, Chen D, Zhang S, Zheng Z. Preparation, characterization and

evaluation of docetaxel-loaded, folate-conjugated PEG-liposomes.

Yakugaku Zasshi. 130(10):1353-9 (2010).

247. Zhai G, Wu J, Yu B, Guo C, Yang X, Lee RJ. A transferrin receptor-

targeted liposomal formulation for docetaxel. J Nanosci Nanotechnol.

10(8):5129-36 (2010).

ANEXOS

Anexo 1: Artículo relacionado

Chitosan-coated lipid nanocarriers for therapeutic applications

F.A. Oyarzun-Ampuero, M. Garcia-Fuentes, D. Torres, M.J. Alonso

Departamento de Farmacia y Tecnología Farmacéutica, Facultad de

Farmacia, Universidad de Santiago de Compostela, 15782-Santiago de

Compostela, Spain

Adapted from: Journal of drug delivery science and technology. (2010). 20

(4): 259-265.

Anexo 1

253

Abstract

This article reports the efforts made over the last two decades by our

group regarding the design of chitosan-coated lipid nanostructures as well as

their potential for two different applications: i) as transmucosal delivery

vehicles for complex macromolecules and ii) as carriers for anticancer drug

delivery. The nanostructures described here share a core coating structure, in

which the core consists of a liquid (Miglyol 812) or a solid (tripalmitin) lipid

surrounded by a chitosan coating. Both polymer-coated nanostructures have

displayed outstanding properties in relation to the favored transport of large

complex molecules (e.g. peptides and vaccines) across the nasal and

intestinal barriers, as well as to facilitate

the intracellular delivery of anticancer drugs into tumor cells.

Keywords: Chitosan; Nanocapsules; Lipid nanoparticles; Transmucosal

peptide delivery; Cancer therapy.

Anexo 1

255

Chitosan-coated lipid nanostructures, rationale and historical

perspective

For better comprehension of our contribution to the development of

chitosan-coated lipid nanocarriers, the reader should refer to the past, more

precisely, to the context of the mid 90s. At that time, lipid nanocarriers such

as submicron nanoemulsions, nanocapsules and solid lipid nanoparticles,

were revealed as interesting systems for the administration of hydrophobic

drugs by different routes [1-3]. Most of these colloidal drug carriers were

characterized by a negative surface charge, which was mainly attributed to

the presence of natural lipidic compounds [3, 4] or to polyester polymers [1,

5]. The presence of this negative surface charge helped prevent the

destabilization of the carriers; however, it also had certain negative

consequences. On the one hand, it promoted the adsorption of cationic

proteins and sodium and calcium ions present in biological fluids, thereby

leading to the neutralization of the surface charge, breakdown of the system

and leakage of the entrapped agents [6]. On the other, these negatively

charged colloidal systems can suffer an electrostatic repulsion with biological

membranes, since they are also negatively charged [6]. In the light of the

above, it was reasonable to believe that positively charged drug carriers

would be favorable in terms of facilitating interactions with the epithelia and

improving the capacity for drug transport [7, 8]. In fact, several authors

proposed the use of positive phospholipid derivates and other cationic

surfactants in the preparation and stabilization of liposomes and submicron

emulsions [9-11]. As an alternative, we proposed a new approach based on

the incorporation of the cationic polysaccharide chitosan (CS) on the surface

of the nanocarrriers simply through ionic interactions between the negatively

charged nanocarrier surface and the polymer [6]. The polysaccharide CS was

chosen to coat these colloidal systems due to its cationic character and

because it showed critical features for drug delivery, including


256

mucoadhesivity and biocompatibility. Indeed, several authors had reported

low or absent signs of toxicity upon CS administration by different routes

[12-13]. Our first attempts conducted with the newly developed CS-coated

lipid nanocarriers focused on the ocular route, in which several problems

such as removal mechanisms, rapid nasolachrymal drainage and non-

productive drug absorption into the systemic circulation constrains the effects

of conventional formulations. With the benefits obtained with these CS-

coated lipid nanocarriers, we can achieve a higher drug concentration in

ocular tissues and improve most pharmacokinetic parameters when compared

to other cationic- coated carriers or to commercial formulations [14]. This

information together with the low ocular toxicity of the nanosystems [14],

inspired us to extend applications of these novel formulations to other

administration routes.

This work aims to review and to summarize our group’s contribution

in aspects concerning the design and in vivo fate of the developed CS-coated

lipid nanocarriers.

Design and characterization of CS-coated lipid nanocarriers

CS nanocapsules

The initially designed CS-coated nanosystems were formed by

promoting the ionic interaction of CS onto the surface of the negatively

charged poly-ε-caprolactone (PεCL) nanocapsules or onto the negatively

charged submicron nanoemulsion [6]. In the first case, CScoated PεCL

nanocapsules were obtained by modifying the method of interfacial

deposition of polyester polymers, proposed by Al Khouri et al. [15]. PεCL

nanocapsules were formed by means of the spontaneous emulsification of a

lipid core (Mygliol and lecithin) in water due to the diffusion of the organic

solvent, in which the polymer and lipids were dissolved [15, 16]. To obtain

CS-coated polyester nanocapsules, the polysaccharide CS was incorporated

Anexo 1

257

in the aqueous phase during the manufacturing process, thus allowing the

adsorption of CS onto the negatively charged surface of the systems [6].

In the second case, CS nanocapsules were prepared using a similar

procedure to the one described above, the only difference being that the PεCL

was eliminated from the formulation. Thus, the method of preparation was

not a polymeric interfacial deposition, but rather a direct electrostatic

interaction between CS and lecithin located in the inner core of the systems

[6]. We also described the possibility of incubating the polysaccharide CS

with the preformed lecithin-containing nanoemulsions for obtaining these

nanocapsules [17].

Characterization by photon correlation spectroscopy of CS-coated

polyester and CS nanocapsules indicated that nanocarriers between 150-300

nm were obtained, showing a monomodal dispersity. Laser doppler

anemometry showed that all systems without CS displayed negative zeta

potential values, while the addition of CS to the aqueous phase resulted in

highly positive values, supporting the successful formation of a CS layer

surrounding the lipid cores. Micrographs obtained by transmission electron

microscopy (TEM), showed spherical structures in all cases (Figure 1).

Figure 1 - Transmission electron micrographs of (a) CS-coated polyester nanocapsules and (b) CS nanocapsules (reproduced with permission from Springer and Editions de Santé, respectively).


258

Considering the hydrophobic nature of the inner components in CS-coated

nanosystems, it was reasonable to postulate that a variety of molecules with

low water solubility could be efficiently encapsulated. The drugs that we

effectively encapsulated in the CS-coated nanosystems included: diazepam

[6], triclosan [18] and docetaxel [19]. Additionally, the cationic CS coating

allowed the adsorption of negatively charged hydrophilic macromolecules

such as the recombinant hepatitis B surface antigen [20].

The presence of the negatively charged lecithin in the oily core of the

nanocapsules further allowed the incorporation of positively charged

hydrophilic molecules such as salmon calcitonin [21]. The peptide was

encapsulated into CS nanocapsules with an efficiency of 44 %, which was

significantly lower than that shown by the uncoated nanoemulsion (> 90 %).

This effect was attributed to a competition between the peptide and the CS,

both positively charged, in their association with the surface of the

nanoemulsion. The encapsulation efficiency was also dependent on the CS

molecular weight. In fact, CS oligomer (~10 kDa) nanocapsules showed

higher encapsulation efficiency (60 %) than medium molecular weight (~100

kDa) CS nanocapsules. This indicates that the CS oligomer coating was not

able to displace salmon calcitonin to the same extent as medium molecular

weight CS.

CS-coated lipid nanoparticles

Solid triglycerides are interesting biomaterials for the production of

nanocarriers due to: i) their excellent biocompatibility [22]; ii) their capacity

to form highly hydrophobic matrices capable of restricting the penetration of

peptidases and other degradative enzymes present in biological fluids or in

the intestinal epithelia, thus protecting the loaded cargo; iii) their capacity to

enhance intestinal drug transport, which has been observed even in the case

Anexo 1

259

of peptidic drugs [23]. Taking these reasons into account, we decided to

prepare CS-coated solid triglyceride nanoparticles as alternative formulations

to CS nanocapsules. More concretely, our aim was to compare the suitability

of the different core structures, based on liquid or solid lipids, for protecting

and promoting the intestinal absorption of peptide drugs. Solid triglyceride

nanoparticles are typically prepared by high-pressure homogenization of

molten lipid mixtures, by formation of microemulsions above the melting

temperature of the triglycerides or by solventemulsification techniques [24].

These preparation techniques allow simple microencapsulation of

hydrophobic molecules such as doxorubicin, paclitaxel, prednisolone or

progesterone, among others [25]. On the other hand, solid triglyceride

matrices present low affinity for hydrophilic macromolecules. Indeed, early

attempts to include proteins and peptides into nanomatrices of solid lipids

were undertaken by solubilizing these molecules in molten lipid mixtures that

were subsequently dispersed as nanometric matrices [26, 27]. These methods

frequently resulted in low protein loadings and potential peptide denaturation

processes arising from contact with the molten lipids [24]. To avoid the use

of high temperatures, we decided to adapt a solvent casting method and more

specifically, a w/o/w doubleemulsion solvent-evaporation technique [28].

The double-emulsion solvent-evaporation method has been applied to the

encapsulation of insulin and salmon calcitonin, leading to moderate/high

encapsulation efficiencies and drug loadings close to 1 % (w/w) [28, 29].

Such encapsulation values represented a significant improvement over

previous attempts based on peptide solubilization in the triglyceride mixture

[27]. Notably, double-emulsion solvent-evaporation techniques have found

application in the encapsulation of other biopharmaceutics such as antigenic

proteins [30]. CS-coated lipid nanoparticles can be prepared by the

deposition of CS on the nanoparticle surface, a process that is triggered by

electrostatic forces. Presence of CS in the external phase of the solvent

emulsification method used for lipid nanoparticle preparation destabilizes the


260

colloidal system. For that reason, our method of CS-coated lipid

nanoparticles preparation comprises first the preparation of the lipid

nanoparticle cores and secondly, the formation of the CS coating by

electrostatic interactions between the anionic cores and polycationic CS [29].

Typical CS-coated nanoparticles prepared by this method present a particle

size between 400 and 600 nm and a positive zeta potential, as confirmed by

photon correlation spectroscopy and laser doppler anemometry. Lipid

nanoparticle composition and nanostructure can be further characterized by

liquid-state NMR techniques, taking advantage of the different relaxation

constants of the triglyceride core and polymeric shell and of the possibility of

performing the analysis over the lipid mixture melting temperature [31, 32].

CS-coated nanostructures for the transmucosal delivery of

macromolecules

The beginning of this decade witnessed large research efforts

dedicated to finding the “holy grail” of transmucosal delivery, a carrier

capable of promoting the oral absorption of protein and DNA-based

medicines [33]. The expectation of realizing such groundbreaking technology

was fuelled by major advances from the late 80s and 90s, that demonstrated

the capacity of nanometric matter to interact with the intestinal epithelium,

ultimately leading to the first proofs-of-principle of their capacity to enhance

the bioavailability of macromolecules delivered by transmucosal routes.

Some of these first key contributions came from the Florence group, which in

a systematic work demonstrated the capacity of submicrometric matter to be

taken up by the intestinal epithelium and to a lesser extent, to be internalized

[34-36]. Simultaneously, the Couvreur group presented the first evidence of

efficient peptide delivery by the oral route using polycyanoacrylate

nanocapsules [37]. This concept of oral delivery of therapeutic

Anexo 1

261

macromolecules was further explored by the Mathiowitz group, this time

using biodegradable, solid-core nanoparticles [38].

During the 90s, our group also studied the capacity of polymeric

nanocarriers to interact with several epithelial barriers. Our results indicated

the capacity of nanocarriers to tightly interact with the corneal, nasal and

intestinal epithelia and even more importantly, that this interaction can lead

to significant improvements in the transmucosal absorption of drugs [39-41].

All of this collected evidence pointed us to a potential opportunity to

apply specifically optimized nanostructures for the transmucosal delivery of

macromolecules. The blueprint of an ideal transmucosal nanocarrier would

be a system capable of protecting the macromolecule of interest from

enzymatic degradation in the administration route [42], but also capable of

enhancing macromolecule transport through biological barriers.

CS is a biocompatible polymer with mucoadhesive and penetration-

enhancing properties, which makes it a promising candidate for nanocarrier

surface modification [43]. Additionally, studies performed by our group have

confirmed that CS-coated nanostructures present improved stability in

biological fluids compared to lipid nanocores [29]. The modification of

nanocarriers with CS applied as a mucoadhesive polymer coating for

transmucosal delivery was reported simultaneously by the Kawashima group

and our own. These works comprised CScoating strategies implemented into

PεCL nanocapsules, poly(lactic-co-glycolic acid) nanoparticles and

liposomes as transmucosal peptide carriers [6, 44-46]. In the next part of the

review, we will summarize our main findings in the field of transmucosal

delivery using the proposed CS-coated lipid nanocarriers. The interaction of

the nanocarriers with mucosal surfaces and the administration of peptidic

drugs by oral and nasal routes will be reviewed. As an alternative application,

the utilization of CS nanocapsules in cancer therapy will be presented.

Interaction of CS-coated nanostructures with mucosal surfaces


262

We had previously reported a study showing the preferential

internalization of CS nanoparticles compared with poly(ethylene glycol)

(PEG)-coated nanoparticles in the Caco-2 cell model [47]. These results were

even more remarkable when the formulations were assayed in the mucus-

secreting cells MTX-E12, suggesting that mucoadhesivity of CS

nanoparticles is an important factor for interacting with mucosal surfaces.

The above findings contrast with those obtained with CS-coated lipid

nanostructures in Caco-2 cells. Here, the amount of associated CS-coated

lipid nanoparticles or CS nanocapsules was similar to those obtained with

PEG-coated nanoparticles or with a nanoemulsion, respectively [17, 48]. A

further goal was to elucidate the possible internalization together with the

intracellular localization of CS-coated lipid nanostructures. For this, CS

nanocapsules were visualized after their incubation with Caco-2 cells and

with a coculture of enterocytes and mucus-secreting cells using confocal laser

scanning microscopy [21]. The results indicated that i) while in Caco-2 cells

nanosystems interacted with a random distribution, in the coculture they

interacted highly and preferentially with the mucus secreting cells; ii) the

systems were not capable of crossing the monolayer and were preferentially

located in the apical region of the cells (Figure 2). The higher interaction

with mucus secreting cells of CS-coated nanosystems was attributed to the

strong mucoadhesive character of the polymer which is related to the

formation of hydrogen and ionic bonds between the positively charged amino

groups of CS and the negatively charged sialic acid residues of mucin

glycoproteins [49]. Consequently, we can infer from this study that the

mucoadhesive character of CS nanocapsules is a determinant factor of their

ability to interact with the intestinal mucosa. Theoretically, this mechanistic

behavior would also be applicable to other CS-coated nanostructures such as

lipid nanoparticles [29]. We have also checked the capacity of our polymer-

coated lipid nanostructures to modify the permeability of the cell monolayers.

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263

In this case, it is known that CS has a capacity to cause a dose-dependent

decrease in transepithelial electric resistance (TEER) [50] while PEG is

assumed to be inert in terms of cellular interaction. In agreement with this,

we observed that PEG-coated lipid cores did not cause a reduction in the

TEER of Caco-2 monolayers in the range of concentrations investigated

(220-330 μg/cm2). In contrast, CS-coated lipid nanostructures induced a

dose-dependent drop in the TEER of Caco-2 monolayers, reaching significant

reductions for the concentration range of 83-330 μg/cm2 [17, 48] (Figure 3).

The values of the TEER reduction were additionally supported by the

observation of enhanced paracellular transport of the macromolecular marker

dextran-Texas Red (Mw = 3000 Da) [48].

However, it should be added that these values were, in the case of

nanocapsules, close to those that comprised cell viability (220 μg/cm2) [17].

Therefore, it could be expected that this change in epithelium permeability

could only be seen when an important amount of the nanocarriers accumulate

in the epithelium. Interestingly, we observed that the normal TEER values

slowly recuperated after the exposure of CS-coated nanostructures.


264

Figure 2 - Confocal scanning microscopy images showing the association of fluorescent nanocapsules (green) with Caco-2 cells and coculture Caco-2/HT29-M6 (E-cadherin in red and nucleus in blue). Caco-2: (A1) Montage of 24 horizontal cross sections illustrating the interaction of fluorescent CS nanocapsules to the cells (step size in z axis of 0.5 2 m.); (A2) confocal xz section showing the accumulation of fluorescent CS nanocapsules in the apical side of the monolayer. Caco-2/HT29-M6 coculture: (B1) Montage of microscopy images showing the association of fluorescent CS nanocapsules with the coculture Caco-2:HT29-M6. (B2) Confocal scanning microscopy xz section showing the accumulation of fluorescent CS nanocapsules (green) in the apical side of the HT29-M6 cells (reprinted with permission from Springer).

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265

Figure 3 - Transepithelial electric resistance (TEER) of Caco-2 monolayers exposed to PEG-coated nanoparticles (1 mg/mL) (◊), CS-coated nanoparticles (1 mg/mL) (s) or their respective controls (HBSS (Hanks’ balanced salt solution) (p); HBSS pH 6.5 (l)) (mean ± SD, n = 3-6) (reprinted with permission from Elsevier). Application of CS-coated nanocarriers for oral peptide delivery – case of study: salmon calcitonin

The efficacy of CS nanocapsules and CS-coated lipid nanoparticles

as oral carriers for peptide delivery was investigated using salmon calcitonin

(sCT) as a model [17, 48]. The reduction of the serum calcium levels after

oral administration of peptide-loaded CS nanocapsules was monitored in rats,

using an aqueous solution and a nanoemulsion containing sCT as controls.

Importantly, as shown in Figure 4, a marked hypocalcemic response was

noted when the peptide was associated with the nanocapsules. The

importance of the CS coating was evident because sCT was ineffective when

administered in the uncoated nanoemulsion. Moreover, this reduction in the

serum calcium levels was maintained for more than 24 h following

administration of the CS-coated nanocapsules. This pronounced and

longlasting hypocalcemic effect led us to speculate that the mucoadhesive

properties of CS might be determinant in facilitating the intestinal absorption

of sCT and in assuring the sustained release of the peptide from the

absorptive epithelium towards the bloodstream. In the same way, sCT-loaded


266

CS-coated lipid nanoparticles administered orally to rats have shown a very

marked and prolonged pharmacological effect, which contrasted with the lack

of response observed for the controls of sCT solution and PEG-coated lipid

nanoparticles [48].

Application of CS-coated nanocarriers for nasal peptide delivery-case of

study: salmon calcitonin

Our research group was the first to explore the potential of CS

nanocapsules to increase the nasal absorption of peptide drugs. Several

strategies had been proposed to enhance the bioavailability of peptides by the

nasal route, with the use of penetration enhancers being a frequent option.

Importantly, a major limitation of the majority of enhancers is related to their

ability for inducing morphological changes on the nasal mucosa and/or

inhibition of the ciliary movement [51, 52]. Among the penetration

enhancers, CS is a special case because, in addition to being a mucoadhesive

material, the penetration enhancing effect of this polymer is reversible;

therefore, it does not compromise the integrity and functionality of the

epithelia [53, 54]. The efficacy of this material in terms of increasing the

nasal absorption of peptides has already been illustrated for insulin,

leuprolide, parathyroid hormone and sCT [54-56]. In our study, the model

peptide sCT was incorporated in the nasally administered formulations and

the serum calcium levels obtained with the CS nanocapsules were compared

with those obtained with the uncoated nanoemulsion and with an aqueous

solution containing or not containing the CS [57]. The results shown in

Figure 5 indicate that the hypocalcemic effect observed after administration

of the colloidal systems (uncoated nanoemulsion and CS nanocapsules) was

significantly greater than that obtained with the other formulations, with

results being clearly better for the CS nanocapsules. Interestingly, the slight

but significant decrease in calcium levels observed for the uncoated

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267

nanoemulsion might be related to the lipids’ absorptionenhancing effect [23,

58] and/or to their drug-protecting properties [59, 60]. The results obtained

with CS nanocapsules compared to the other formulations, highlight the

critical role of CS in enhancing the transport of the associated drug and

consequently underline the potential of this polymer for nasal peptide

delivery. We have also prepared and tested CS nanocapsules for nasal

vaccination. In that case, the recombinant hepatitis B surface antigen was

adsorbed ionically onto the surface of the nanocapsules. These systems were

capable of inducing an effective protective response against the disease [20,

61]. This subject will be extensively reviewed in the article “From single-

dose vaccine delivery systems to nanovaccines” published in this same issue.

Figure 4 - Serum calcium levels after oral administration in rats of CS nanocapsules as well as the control nanoemulsion and an aqueous solution of sCT (mean ± SE; n = 6). sCT solution;

nanoemulsion; CS nanocapsules. *Statistically significant differences from sCT solution (p < 0.05) (reprinted with permission from Elsevier).


268

Figure 5 - Serum calcium levels after nasal administration in rats of salmon calcitonin (sCT, dose: 15 IU/kg) in aqueous solution (with or without CS) or encapsulated in the control nanoemulsion or in CS nanocapsules; (mean ± SE; n = 6). sCT solution; X sCT solution + CS; nanoemulsion; CS nanocapsules. *Significantly different from salmon sCT solution (p < 0.05). #Significantly different from nanoemulsion (p < 0.05) (reproduced with permission from Editions de Santé). CS nanocapsules for anticancer drug delivery

The advances in nanomedicine are especially well perceived in

cancer therapy, mainly because the efficacy of current treatments is still very

limited [62]. The main problems related to conventional cancer therapies

include: unspecific biodistribution, suboptimal cell internalization and the

toxicity of standard excipients required for drug solubilization. In this

context, nanocapsule technology emerges as apromising approach for the

formulation of anticancer drugs, offering the possibility of facilitating the

accumulation of the drug in the tumor tissue by the well-known enhanced

permeability and retention effect (EPR) [63]. Nanocapsules may also

improve the cellular internalization of the drug because of its ability to inhibit

the multi-drug resistance barrier expressed in many kinds of cancers [64] and

allowing the incorporation of hydrophobic drugs directly into the

formulation, thus limiting the toxicity of excipients while protecting the drug

from biological fluids [65].

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Taxanes (paclitaxel, docetaxel) have established themselves as an

important class of currently available antitumor drugs. They have greatly

contributed to the improvement in cancer patient survival and have proven

clinical efficacy against a wide range of solid tumors, such as advanced

breast, ovarian or non-small cell lung cancer [66-68]. Despite their positive

therapeutic features, taxanes suffer from such drawbacks as aqueous

insolutibilty and dose-limiting toxicities at the clinically administered doses,

especially related to the solubilizing solvents and surfactants included in their

marketed formulations. Importantly, the potential of nanoformulated taxanes

to solve this inherent toxicity was reinforced by the Food and Drug

Administration’s approval of Abraxane, consisting of nanoparticles

containing albumin-bound paclitaxel [69].

CS nanocapsules developed by our research group were proposed as

carriers for the taxane drug docetaxel with the aim of reducing the side

effects of the free drug and improving docetaxel’s efficacy by tumor targeting

[19]. In this study oligomers of CS were chosen because a low molecular

weight of the polymer was considered safer to be administered by

intravenous route.

Docetaxel-loaded CS nanocapsules were incubated with MCF7

(breast tumor cell line) and A549 (lung tumor cell line) cells for 24 and 48 h,

showing an effect on cell proliferation at 24 h that was significantly greater

than that of free docetaxel in both cell lines. This information was

corroborated by the lower growth inhibition 50 % (GI50) values obtained for

the loaded nanocapsules compared to the free drug in both cell cultures

(Table I). Importantly, in Table I is also possible to appreciate that blank CS

nanocapsules had no significant effects in the cell growth.


270

Interestingly, after 48 h of incubation, the effects on cell proliferation

observed for docetaxel-loaded nanocapsules and free docetaxel were similar,

which suggests that the differential effect found at 24 h was due to an

accelerated uptake of the docetaxel-loaded nanocarriers. In order to provide

evidence of the uptake intensity of the CS nanocapsules, a fluorescent dye

was encapsulated and its interaction was evaluated at 2 h by flow cytometry

in MCF-7 and A549 cell lines. Figure 6 shows that the encapsulated

fluorescent dye interacted with almost every cell, while the non encapsulated

dye remained mainly excluded from this interaction. This improved uptake

could be related to a favorable interaction of the CS coating with the cancer

cells, as it was also found with CS nanocapsules in different cell types such

as those of corneal epithelium [70].

More recently, a comparative efficacy study with docetaxel-loaded

CS-nanocapsules and a control docetaxel formulation (Taxotere) was

performed in a A549 tumor xenograf model in mice, by administering an

intratumoral dose of 9 mg/kg, each four days, for a complete dose of 27

mg/kg. In this work, we have shown that the efficacy of docetaxel-loaded

CS-nanocapsules was comparable to that of the classical docetaxel

formulation [71]. Moreover, by analyzing the effect of each single injection

of both docetaxel formulations in the tumor size, it was found that free

docetaxel has a faster effect but docetaxel-loaded CS nanocapsules can

continue their antiproliferative effect for longer periods of time. Besides this

hypothetical advantage which needs to be corroborated, CS oligomer

nanocapsules exhibit the great advantage to avoid the use of Tween 80 for the

solubilization and formulation of docetaxel, this vehicle being responsible for

severe side effects. Therefore, CS oligomer nanocapsules can be proposed as

a new drug delivery system for docetaxel, although toxicological and

biodistribution studies need to be undertaken to support the potential of this

formulation.

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Table I - GI50 (growth inhibitory 50, drug concentration resulting in a 50 % reduction in absorbance in control cells) in MCF7 and A549 cells for blank CS nanocapsules, free docetaxel, and docetaxel-loaded CS nanocapsules (mean ± SD; n = 4) (Reproduced with permission from ACS publications).

nd: non determined; none of the concentrations tested resulted in a 50 % reduction of the absorbance.

Figure 6 - Uptake studies of fluorescent CS nanocapsules assessed by flow cytometry in MCF7 and A549 cells. Percentage of stained cells after a 2 h incubation with fluorescein-DHPE CS nanocapsules (black bars) or free fluorescein-DHPE (white bars). Mean values of three independent experiments. (Reproduced with permission from ACS publications).


272

5. Main remarks

CS-coated lipid nanostructures are a versatile tool suitable for

encapsulating lipophilic or hydrophilic drugs with a considerable efficiency

and capable of exhibiting sustained-release functions at the site of delivery or

action. These CS lipid nanocarriers have shownpotential as transmucosal

carriers for the delivery of large complex molecules and also as intracellular

drug delivery vehicles. Besides the capacity of these nanocarriers to protect

sensitive molecules in their lipid core, the presence of the CS coating has

been identified as a critical parameter for their efficacy. This coating is

responsible for the interaction of the nanostructures with the epithelial and

cellular barriers. More specifically, these interactions are known to favor the

residence of the nanostructures in the intestinal epithelium and facilitate the

rapid uptake into the cancer cell lines. A proof-of-principle has been

described for drugs such as salmon calcitonin and docetaxel. Current

experiments are aimed at evidencing the clinical relevance of these findings

and identifying potential drug/vaccine candidates which could greatly benefit

from these findings.

References

1. Calvo P., Vila-Jato J.L., Alonso M.J. - Comparative in vitro evaluation

of several coloidal systems, nanoparticles, nanocapsules, and

nanoemulsions, as ocular drug carriers. - J. Pharm. Sci., 85, 530-536,

1996.

2. Losa C., Marchal-Heussler L ., Orallo F., Vila-Jato J.L. Alonso M.J. -

Desing of new formulations for topical ocular administration: polymeric

nanocapsules containing metipranolol. - Pharm. Res., 10, 80-87, 1993.

3. Müller R.H., Maassen S., Weyhers H., Mehner T. – Phagocityc uptake

and cytotoxicity of solid lipid nanoparticles (SLN) sterically stabilized

Anexo 1

273

with poloxamine 908 and poloxamer 407. - J. Drug. Target., 4 (3), 161-

170, 1996.

4. Benita S., Levy M.Y. - Submicron emulsions as colloidal drug carriers

for intravenous administration: comprehensive physicochemical

characterization. - J. Pharm. Sci., 82 (11), 1069-1079, 1993.

5. Guterres S.S., Fessi H., Barratt G., Puisieux F., Devissaguet J.P. -

Poly(D,L-lactide) nanocapsules containing non-steroidal anti-

inflammatory drugs: gastrointestinal tolerance following intravenous

and oral administration. - Pharm Res., 12 (10), 1545-1547, 1995.

6. Calvo P., Vila-Jato J.L., Alonso M.J. - Development of positively

charged coloidal drug carriers: chitosan-coated polyester nanocapsules

and submicron-emulsions. - Colloid. Polym. Sci., 275, 46-53, 1997.

7. Meisner D., Pringle J., Mezei M. - Liposomal ophthalmic drug delivery.

III. Pharmacodynamic and biodisposition studies of atropine. - Int. J.

Pharm., 55, 105-113, 1989.

8. Robinson J.R. - Ocular drug delivery. Mechanism(s) of corneal drug

transport and mucoadhesive delivery systems. – STP Pharma, 5, 839-

846, 1989.

9. Guo L.S.S., Radhakrishnan R., Redemann C.T. - Adhesion of

positivively charged liposomes to mucosal tissues. - J. Liposomal Res.,

1, 319-337, 1989.

10. Lee H.V.L., Carson L.W. - Ocular disposition of inulin from single &

multiple doses of positively charged multilamellar liposomes: evidence

for alterations in tear dynamics and ocular surface characteristics. - J.

Ocular Pharmacol., 2, 353-364, 1986.

11. Elbaz E., Zeevi A., Klang S., Benita S. - Positively charged submicron

emulsions- a new type of colloidal drug carrier. - Int. J. Pharm., 96, R1-

R6, 1993.


274

12. Weiner M.L. - In: Advances in Chitin and Chitosan, C.J. Brine., P.A.

Sandford., J.P. Zikakis Eds., Elsevier Science Publishers Ltd., London,

1993, pp. 663-672.

13. Hirano S., Seino H., Akiyama Y., Nonaka I. - In: Progress in

Biomedical Polymers, C.G. Gebelein., R.L. Dunn Eds., Plenum Press,

New York, 1990, pp. 283-289.

14. Calvo P., Vila-Jato J.L., Alonso M.J. - Evaluation of cationic polymer-

coated nanocapsules as ocular drug carriers. - Int. J. Pharm., 153, 41-50,

1997.

15. Al-Khouri F.N., Roblot-Treupel L., Fessi H. - Development of a new

process for the manufacture of polyisobutylcyanoacrylate nanocapsules.

- Int. J. Pharm., 28, 125-132, 1986.

16. Fessi H., Puisieux F., Devissaguet J.P., Ammoury N., Benita S. -

Nanocapsule formation by interfacial polymer deposition following

solvent deplacement. - Int. J. Pharm., 55, 25-28, 1989.

17. Prego C., Garcia M., Torres D., Alonso M.J. – Transmucosal

macromolecular drug delivery. - J. Control Release., 101, 151-162,

2005.

18. Maestrelli F., Mura P., Alonso M.J. - Formulation and characterization

of triclosan sub-micron emulsions and nanocapsules. - J.

Microencapsul., 21 (8), 857-64, 2004.

19. Lozano M.V., Torecilla D., Torres D., Vidal A., Dominguez F., Alonso

M.J. - Highly efficient system to deliver taxanes into tumor cells:

docetaxel-loaded chitosan oligomer colloidal carriers. - Biomacromol.,

9, 2186-2193, 2008.

20. Vicente S., Sanchez A., Diaz B., Gonzalez A., Alonso M.J. -

Polysaccharide based nanocarriers for intranasal vaccination: application

to rHBs antigen. - EUFEPS Workshop: Opportunities and Challenges in

Vaccine Delivery, Geneva, 15-17 September 2008, EUFEPS, p. 35.

Anexo 1

275

21. Prego C., Fabre M., Torres D., Alonso M.J. - Efficacy and mechanism

of action of chitosan nanocapsules for oral peptide delivery. - Pharm

Res., 23, 549-56, 2006.

22. Schöler N., Hahn H., Muller R.H., Liesenfeld O. - Effect of lipid matrix

and size of solid lipid nanoparticles (SLN) on the viability and cytokine

production of macrophages. - Int. J. Pharm., 231, 167-176, 2002.

23. Muranishi S. - Absorption enhancers. - Crit. Rev. Therap. Drug Carrier

Systems, 7, 1-33, 1990.

24. Almeida A.J., Souto E. - Solid lipid nanoparticles as a drug delivery

system for peptides and proteins. - Adv. Drug Deliv. Rev., 59, 478-490,

2007.

25. Wissing S.A., Kayser O., Müller R. - Solid lipid nanoparticles for

parenteral drug delivery. - Adv. Drug Deliv. Rev., 56, 1257-1272, 2004.

26. Morel S., Ugazio E., Cavalli R., Gasco M.R. - Thymopentin in solid

lipid nanoparticles. - Int. J. Pharm., 132, 259-261, 1996.

27. Almeida A.J., Runge S., Muller R.H. - Peptide-loaded solid lipid

nanoparticles (SLN): Influence of production parameters. - Int. J.

Pharm., 149, 255-265, 1997.

28. Garcia-Fuentes M., Torres D., Alonso M.J. - Design of lipid

nanoparticles or the oral delivery of hydrophilic macromolecules. -

Colloid Surf. B-Biointerfaces, 27, 159-168, 2003.

29. Garcia-Fuentes M., Alonso M.J., Torres D. - New surface-modified lipid

nanoparticles as delivery vehicles for salmon calcitonin. - Int. J. Pharm.,

296, 122-132, 2005.

30. Saraf S., Mishra D., Asthana A., Jain R., Singh S., Jain N.K. - Lipid

microparticles for mucosal immunization against hepatitis B. - Vaccine,

24, 45-56, 2006.

31. Garcia-Fuentes M., Torres D., Martin-Pastor M., Alonso M.J. -

Application of NMR spectroscopy to the characterization of PEG-

stabilized lipid nanoparticles. - Langmuir, 20, 8839-8845, 2004.


276

32. Garcia-Fuentes M., Alonso M.J., Torres D. - Design and

characterization of a new drug nanocarrier made from solid-liquid lipid

mixtures. - J. Colloid. Interf. Sci., 285, 590-598, 2005.

33. Csaba N., Garcia-Fuentes M., Alonso M.J. - The performance of

nanocarriers for transmucosal drug delivery. - Expert. Opin. Drug

Deliv., 3, 463-478, 2006.

34. Jani P., Halbert G.W., Langridge J., Florence A.T. - The uptake and

translocation of latex nanospheres and microspheres after oral

administration to rats. - J. Pharm. Pharmacol., 41, 809-812, 1989.

35. Florence A.T. - The oral absorption of micro- and nanoparticulates:

neither exceptional nor unusual. - Pharm. Res., 14, 259-266, 1997.

36. Florence A.T., Hussain N. - Transcytosis of nanoparticle and dendrimer

delivery systems: evolving vistas. - Adv. Drug Deliv. Rev., 50, S69-89,

2001.

37. Damgé C., Michel C., Aprahamian M., Couvreur P. - New approach for

oral adiminstration of insulin with polyalkylcyanoacrylate nanocapsules

as drug carrier. - Diabetes, 37, 246-251, 1988.

38. Mathiowitz E., Jacob J.S., Jong Y.S., Carino G.P. – Biologically

erodable microsphere as potential oral drug delivery system. - Nature,

386, 410-414, 1997.

39. Calvo P., Alonso, M.J., Vila-Jato J.L., Robinson J.R. – Improved ocular

bioavailability of indomethacin by novel ocular drugs carriers. - J.

Pharm. Pharmacol., 48 (11), 1147-52, 1996.

40. Tobio M., Gref R., Sanchez A., Langer R., Alonso M.J. – Stealth PLA-

PEG nanoparticles as protein carriers for nasal administration. - Pharm.

Res., 15, 270-275, 1998.

41. Tobio M., Sanchez A., Vila A., Soriano I., Évora C., Vila-Jato J.L.,

Alonso M.J. - The role of PEG on the stability in digestive fluids and in

vivo fate of PEG-PLA nanoparticles following oral administration. -

Colloid. Surf. B-Biointerfaces, 18, 315-323, 2000.

Anexo 1

277

42. Lee Y.H., Sinko P.J. - Oral delivery of salmon calcitonin. - Adv. Drug

Deliv. Rev., 42, 225-238, 2000.

43. Janes K.A., Calvo P., Alonso M.J. - Polysaccharide colloidal particles as

delivery systems for macromolecules. - Adv. Drug Deliv. Rev., 47, 83-

97, 2001.

44. Kawashima Y., Yamamoto H., Takeuchi H., Kuno Y. – Mucoadhesive

DL-lactide/glycolide copolymer nanospheres coated with chitosan to

improve oral delivery of elcatonin. - Pharm. Dev. Technol., 5, 77-85,

2000.

45. Takeuchi H., Yamamoto H., Niwa T., Hino T., Kawashima Y. - Enteral

absorption of insulin in rats from mucoadhesive chitosan-coated

liposomes. - Pharm. Res., 13, 896-901, 1996.

46. Takeuchi H., Matsui Y., Yamamoto H., Kawashima Y. – Mucoadhesive

properties of carbopol or chitosan-coated liposomes and their

effectiveness in the oral administration of calcitonin to rats. - J. Control.

Release, 86, 235-242, 2003.

47. Behrens I., Pena A.I., Alonso M.J., Kissel T. - Comparative up-take

studies of bioadhesive and non-bioadhesive nanoparticles in human

intestinal cell lines and rats: the effect of mucus on particle adsorption

and transport. - Pharm. Res., 19, 1185-93, 2002.

48. Garcia-Fuentes M., Prego C., Torres D., Alonso M.J. - A comparative

study of the potential of solid triglyceride nanostructures coated with

chitosan or poly(ethylene glycol) as carriers for oral calcitonin delivery.

- Eur. J. Pharm. Sci., 25, 133-43, 2005.

49. Rossi S., Ferrari F., Bonferoni M.C., Caramella C. – Characterization of

chitosan hydrochloride-mucin interaction by means of viscosimetric and

turbidimetric measurements. - Eur. J. Pharm. Sci., 10, 251-257, 2000.

50. Borchard G., Lueben H, De Boer A.G., Verhoef J.C., Lehr C.M.,

Junginger H.E. - The potential of mucoadhesive polymers in enhancing

intestinal peptide drug absorption. III. Effects of chitosan-glutamate and


278

carbomer on epithelial tight junctions in vitro. - J. Control Rel., 39, 131-

138, 1996.

51. Merkus F.W.H.M., Schipper N.G.M., Hermens W.A.J.J., Romeijn S.G.,

Verhoef J.C. - Absorption enhancers in nasal drug delivery: efficacy and

safety. - J. Control. Release, 24, 201- 208, 1993.

52. Marttin E., Verhoef J.C., Romeijn S.G., Merkus F.W.H.M. – Effects of

absorption enhancers on rat nasal epithelium in vivo: release of marker

compounds in the nasal cavity. - Pharm. Res., 12, 1151-1157, 1995.

53. Tengamnuay P., Sahamethapat A., Sailasuta A., Mitra A.K. - Chitosan

as nasal absorption enhancers of peptides: comparison between free

amine chitosans and soluble salts. - Int. J. Pharm., 197, 53-67, 2000.

54. Illum L., Farraj N.F., Davis S.S. - Chitosan as a novel nasal delivery

system for peptide drugs. - Pharm. Res., 11, 1186-1189, 1994.

55. Illum L. - Nasal drug delivery-possibilities, problems and solutions. - J.

Control. Release, 87, 187-198, 2003.

56. Sinswat P., Tengamnuay P. - Enhancing effect of chitosan on nasal

absorption of salmon calcitonin in rats: comparison with hydroxypropyl-

and dimethyl-ß-ciclodextrins. - Int. J. Pharm., 257, 15-22, 2003.

57. Prego C., Torres D., Alonso M.J. - Chitosan nanocapsules: A new

carrier for nasal peptide delivery. - J. Drug Del. Sci. Tech., 16, 331-337,

2006.

58. Mitra R., Pezron I., Chuw A., Mitra A.K. - Lipid emulsions as vehicles

for enhanced nasal delivery of insulin. - Int. J. Pharm., 205, 127-134,

2000.

59. Lowe P.J., Temple C.S. - Calcitonin and insulin in

isobutylcyanoacrylate nanocapsules: Protection against proteases and

effect on intestinal absorption. - J. Pharm. Pharmacol., 46, 547-552,

1994.

Anexo 1

279

60. Yoo H.S., Park T.G. - Biodegradable nanoparticles containing protein-

fatty acid complexes for oral delivery of salmon calcitonin. - J. Pharm.

Sci., 93, 488-495 2004.

61. Csaba N., Garcia-Fuentes M., Alonso MJ. - Nanoparticles for nasal

vaccination. - Adv. Drug. Deliv. Rev., 61, 140-57, 2009.

62. Tanaka T., Decuzzi P., Cristofanilli, Sakamoto J.H., Tasciotti E.,

Robertson F.M., Ferrari M. - Nanotechnology for breast cancer therapy.

- Biomed. Microdevices, 11, 49-63, 2009.

63. Iyer A.K., Khaled G., Fang J., Maeda H. - Exploiting the enhanced

permeability and retention effect for tumor targeting. – Drug Discov.

Today, 11, 812-818, 2006.

64. Garcion E., Lamprecht A., Heurtault B., Paillard A., Aubert- Pouessel

A., Denizot B., Menei P., Benoit J.P. - A new generation of anticancer,

drug-loaded, colloidal vectors reverses multidrug resistance in glioma

and reduces tumor progression in rats. - Mol. Cancer Ther., 5, 1710-

1722, 2006.

65. Couvreur P., Barratt G., Fattal E., Legrand P., Vauthier C. –

Nanocapsule technology: A review. - Crit. Rev. Ther. Drug Carrier

Syst., 19, 99-134, 2002.

66. Twelves C. - Docetaxel weekly with metastatic breast cancer. -

Onkologie, 30, 407-408, 2007.

67. Ferrandina G., Ludovisi M., De Vincenzo R., Salutari V., Lorusso D.,

Colangelo M., Prantera T., Valerio M.R., Scambia G. - Docetaxel and

oxaliplatin in the second-line treatment of platinum-sensitive recurrent

ovarian cancer: a phase II study. - Ann. Oncol., 18, 1348-1353, 2007.

68. Jiang Z., Yan W., Ming J., Yu Y. - Docetaxel weekly regimen in

conjunction with RF hyperthermia for pretreated locally advanced non-

small cell lung cancer: a preliminary study. - BMC Cancer, 7, 189-193,

2007.


280

69. Green M. R., Manikhas G. M., Orlov S., Afanasyev B., Makhson A. M.,

Bhar P., Hawkins M. J. - Abraxane, a novel Cremophorfree, albumin-

bound particle form of paclitaxel for the treatment of advanced non-

small-cell lung cancer. - Ann. Oncol., 17, 1263-1268, 2006.

70. De Campos A.M., Sanchez A., Gref R., Calvo P., Alonso M.J. - The

effect of a PEG versus a chitosan coating on the interaction of drug

coloidal carriers with the ocular mucosa. - Eur. J. Pharm. Sci., 20, 73-81,

2003.

71. Lozano M.V., Torecilla D., Lallana E., Vidal A., Fernandez-Megía E.,

Riguera R., Dominguez F., Alonso M.J., Torres D. – Chitosan

Nanocapsules for active tumor targeting. - Proceedings of the 7th World

Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical

Technology, Malta, 8-11 March 2010.

Acknowledgements

The studies reported in this review were performed in our laboratory

and have been supported by grants from the Ministry of Science and

Technology (Consolider Nanobiomed, CSD 2006-00012) and the Xunta de

Galicia (PGIDIT 08CSA045209PR), Spain. Felipe Oyarzun-Ampuero was

granted a CONICYT scholarship.

Manuscript

Received 7 April 2010, accepted for publication 28 May 2010.

Anexo 2: Artículo relacionado

A new drug nanocarrier consisting of polyarginine and hyaluronic acid

F. A. Oyarzun-Ampueroa, F.M. Goycooleaa, D. Torresa, M.J. Alonsoa

Department of Pharmacy and Pharmaceutical Technology, Faculty of

Pharmacy 15782, University of Santiago de Compostela, Spain.

Adapted from: European Journal of Pharmaceutics and Biopharmaceutics.

(2011). 79(1): 54-57.

Anexo 2

283

Abstract

The purpose of this study was to produce and characterize a variety

of nanostructures comprised of the polyaminoacid polyarginine (PArg) and

the polysaccharide hyaluronic acid (HA) as a preliminary stage before

evaluating their potential application in drug delivery. PArg was combined

with high- or low-molecular-weight HA (HMWHA or LMWHA,

respectively) to form nanoparticles by simply mixing polymeric aqueous

solutions at room temperature. The average size of the resulting nanocarriers

was between 116 and 155 nm, and their zeta potential value ranged from

+31.3 to -35.9 mV, indicating that the surface composition of the particle

could be conveniently modified according to the mass ratio of the polymers.

Importantly, the systems prepared with HMWHA remained stable after

isolation by centrifugation and in conditions that mimic the physiological

medium, whereas particles that incorporated LMWHA were unstable.

Transmission electron microscopy showed that the nanostructures made with

HMWHA were spherical. Finally, the systems were stable for at least three

months at storage conditions (4°C).

Polyarginine

Hyaluronic Acid

GRAPHICAL ABSTRACT

"Nanoparticles can be obtained either with a positive (Polyarginine coating) or a negative surface (Hyaluronate coating)"

"A new drug nanocarrier consisting of a Polyarginine and Hyaluronic Acid matrix was characterized"

Keywords: Nanoparticles; hyaluronic acid; polyarginine; drug delivery;

complexes.

Anexo 2

285

Introduction

Because of their unique size and surface characteristics,

nanostructures have emerged as a platform for the delivery of

macromolecules and low-molecular-weight drugs for the treatment of a

variety of diseases. In nanomedicine, there is a need for suitable biomaterials

for optimization of the interaction/transport of drug carriers to target tissues.

Concerning their characteristics, glycosaminoglycan hyaluronic acid (HA)

and polyaminoacid polyarginine (PArg) are molecules used for the transport

and biorecognition of nanomedicines.

HA is a natural non-toxic mucoadhesive polysaccharide that is

negatively charged and biodegradable. It is widely distributed throughout the

human body but primarily resides in connective tissue, eyes, intestine, and

lungs. Importantly, the overexpression in a variety of tissues of the CD-44

receptor, the endogenous ligand for HA, makes this biopolymer a viable

candidate for specific targeting. Several studies have tested the efficacy of

nanosystems based on HA using various applications, namely gene delivery

[1], cancer [2], and asthma [3] among others.

In turn, polyaminoacids are promising tools for the development of

drug delivery systems, mainly due to their safety profile. These molecules are

structurally similar to polypeptides and are thus degraded by human

enzymes; their accumulation within the organism is minimal. Interestingly,

the cationic PArg is able to translocate through cell membranes and facilitate

the uptake of molecules associated with polyaminoacid [4]. This interesting

feature of PArg has been exploited to harness drug delivery systems used for

gene therapy [5], protein/vaccine delivery [6], and cancer [7]. Furthermore,

PArg enhances the absorption of hydrophilic compounds across the nasal

epithelium [8], a property that may be utilized for mucosal drug delivery.

The nanoparticles preparation method can affect its pharmacological

and functional properties. Some procedures include the use of organic


286

solvents or covalent cross-linkers that can compromise the safety of the

formulation. Other preparation protocols use stringent processes, such as high

temperature or sonication, which may destroy or alter drug bioactivity. In

addition, nanoparticle stability in conditions that mimic biological medium or

during long-term storage is a key factor that may determine the success of the

formulation.

The aim of the present work was to develop and characterize a

variety of PArg-containing nanostructures that were formed by the

combination of the polyaminoacid with HA, wich have also addressed the

possible influence of the molecular weight of HA on the physicochemical

and stability characteristics of the systems by using a high and a low-

molecular-weight hyaluronic acid (HMWHA or LMWHA, respectively).

These particles were formed using an extremely mild and simple procedure

that involves mixing two aqueous phases at room temperature. The prepared

nanostructures could be applied in different fields considering the established

properties such as mucoadhesivity and cell- penetrating capacity of the

constituent polymers. Of note, a recent study by Kim et al. reported the

formation of a nanosystem composed of HA (19 kDa), PArg (15-70 kDa),

and small interference RNA (siRNA) [9]. Such study focused on the

biological behavior of siRNA-loaded nanosystems and not on the

physicochemical and technological parameters of the unloaded nanoparticles.

Anexo 2

287


Chemicals

HMWHA (Mw ~165 kDa) was a gift from Bioiberica (Barcelona,

Spain). LMWHA (Mw ~29 kDa) was purchased from Inquiaroma

(Barcelona, Spain), and polyarginine (PArg, Mw ~5-15 kDa) was purchased

from Sigma Aldrich (Madrid, Spain). All other reagents were of the highest

analytical grade. MilliQ water was used for experimentation.

Preparation of nanoparticles

Nanoparticles were prepared by mixing HA and PArg aqueous

solutions. Briefly, 4.5 mL of an aqueous solution containing HA (0.44-2.67

mg/mL) was added to 4.5 mL of a solution containing PArg (0.53 mg/mL) by

stirring at room temperature. For isolation, 1 mL of the nanoparticles was

transferred to Eppendorf tubes and centrifuged (16000×g, 30 min, 25 º C) in

20 μL of a glycerol bed. Supernatants were discarded, and the nanoparticles

were resuspended in water by vigorous shaking. It is thought that the

nanosystems were formed by electrostatic interactions between the positively

charged amino group of the guanidine moiety on the PArg and the negatively

charged carboxylate groups with the HA.

Physicochemical characterization of nanoparticles


by photon correlation spectroscopy and laser Doppler anemometry using a

Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, United Kingdom),

MilliQ water was used as solvent. Each batch was analyzed in triplicate.

Morphological examination of the nanoparticles was carried out by

transmission electron microscopy (TEM) (CM12 Phillips, Eindhoven,


288

Netherlands). The samples were stained with 1% (w/v) phosphotungstic acid

for 10 s, immobilized on copper grids with Formvar® and dried overnight

before TEM analysis.

Stability of nanoparticles

Nanoparticle formulations were prepared and centrifuged in the

presence of glycerol. The stability of the nanoparticles was evaluated

according to size and precipitation in phosphate-buffered saline (PBS, pH

7.4) at 37 °C and in MilliQ water at 4 °C.

The composition of PBS was as follows: 137 mM NaCl, 2.7 mM KCl, 1.4

mM NaH2PO4 and 1.3 mM Na2HPO4.


Table 1a and b show the mass ratio, charge [HA]/[PArg] ratio, size,

polydispersity index and zeta potential of the tested formulations prepared

with either HMWHA or LMWHA, respectively. Our data show that when the

[HA]/[PArg] charge ratio was lower than 0.975, nanostructures with a

positive zeta potential were obtained, indicating that the surface of these

systems is mainly composed of positively charged PArg. This feature is

attributed to excess of PArg (relative to that of HA) in the formulation.

Consequently, when the [HA]/[PArg] charge ratio increased to 0.975 and

higher, inversion of the zeta potential values was observed, indicating that the

nanocarrier surface was now shielded by excess of HA, which bears a

negative charge. It can be appreciated that the zeta potential values (and

average size) are not further modified beyond this ratio. This indicates that

HA is incorporated into the nanoparticles up to saturation limit while surplus

HA remains unassociated in solution and is in agreement with yield studies

(data not shown).

Anexo 2

289

Table 1: Physicochemical properties of the nanocarriers prepared with different ratios of HMWHA-PArg (a) or LMWHA-PArg (b) and evaluated in MilliQ water. (mean ± S.D., n=3). a)

Mass ratio HMWHA-

PArg

Charge ratio [HA]/[PArg] Size (nm) Polydispersity

index Zeta potential

(mV)

2-2.4 0.325 128 ± 8 0.2-0.3 +31.3 ± 1 4-2.4 0.65 136 ± 16 0.1-0.2 +25.3 ± 4 6-2.4 0.975 154 ± 7 0.1-0.2 -32.5 ± 5 8-2.4 1.3 150 ± 7 0.1-0.2 -33.4 ± 4 10-2.4 1.625 147 ± 7 0.1-0.2 -35.9 ± 5 12-2.4 1.95 155 ± 7 0.1-0.2 -33.8 ± 4

b) Mass ratio LMWHA-

PArg

Charge ratio [HA]/[PArg] Size (nm) Polydispersity

index Zeta potential

(mV)

2-2.4 0.325 Not formed Not formed Not formed 4-2.4 0.65 131 ± 26 0.1-0.2 +25 ± 1 6-2.4 0.975 172 ± 18 0.1-0.2 -19 ± 1 8-2.4 1.3 139 ± 9 0.1-0.2 -27 ± 3 10-2.4 1.625 137 ± 13 0.1-0.2 -31 ± 3 12-2.4 1.95 146 ± 13 0.1-0.2 -33 ± 3

Alteration of the surface charge of the nanocarriers as a function of

the polymer ratio allows optimization of the surface composition (and,

presumably, the biological behavior) to interact with targets that have

affinity for PArg or HA. Additionally, knowledge of the relative contribution

of each charged species to the nanosystems may allow nanoparticle

customization for the incorporation of positively or negatively charged drug

molecules.


290

The transmission electron micrographs indicated that each formulation was

reasonable spherical, which is in agreement with previous works describing a

spherical shape of nanoparticles made with either natural or synthetic

polymers [3]. Figure 1 shows a micrograph of a formulation with a

[HMWHA]/[PArg] charge ratio of 1.3.

Figure 1: Transmission electron micrograph of HMWHA-PArg nanocarriers; HA-PArg charge ratio= 1.3.

Importantly, characterization of HMWHA-containing systems was

conducted after isolation of the nanocarriers by centrifugation, whereas

characterization of LMWHA-containing systems was performed without

isolation. The reason for that was because the latter nanoparticles were

unstable during the centrifugation process (the lower tested conditions of

centrifugation were 500g during 30 min) as evidenced by the disappearance

of the turbidity of the system and by photon correlation spectroscopy (PCS)

size measurements. The systems containing HMWHA were effectively

isolated at 16000g during 30 min, maintaining the same characteristics that

the non-isolated formulations had. As shown in Table 1a and b, at a charge

ratio of 0.325, nanosystems were obtained that incorporated HMWHA,

whereas those containing LMWHA were not formed. In addition, the ϛ values

Anexo 2

291

of the LMWHA-containing systems were generally lower than those

containing HMWHA systems. Thus, fewer charged species were located on

the surface of the low-molecular-weight polymer surface. Greater charge

compensation for systems that have low-molecular-weight species may also

account for the lower ϛ values.

Hence, we postulate that the observed differences among

nanostructures comprising HMWHA or LMWHA are probably due to the

preferential organization of the polymers, particularly at the nanoparticle

surface. Variance in particle assembly may also account for the instability of

LMWHA nanosystems during centrifugation. The formation of local regions

with a large number of consecutive associated residues (i.e., greater

cooperativity) of both polyelectrolytes in the HMWHA-containing systems

may explain its superior stability during centrifugation in comparison with

LMWHA-containing systems, which presumably have lower cooperativity.

These regions, in which charges are compensated, become more hydrophobic

due to their neutral character and, hence, they are expected to lie within the

inner core of the nanostructure. This distribution would provide the systems

with more stability. A similar interpretation was offered in a previous work

studying the behavior of hybrid nanoparticles of chitosan and alginate (of

various molecular weight) cross-linked with tripolyphosphate [10]. It is

important to point out that the ability to isolate formulations by simple

centrifugation avoids tedious time-consuming procedures, such as those

performed by Kim et al. [9], whose developed nanosystems were isolated by

dialysis over 2 days.

Colloidal stability under physiological conditions is a crucial

characteristic for successful biomedical application of the nanosystems.

Therefore, we investigated the stability of the systems composed of

HMWHA and LMWHA in PBS at pH 7.4 and 37 °C. Figure 2 shows the

stability profiles of the HMWHA-containing systems. The majority of the

formulations were stable for at least 2 h, indicating that the stability of the


292

nanosystems was maintained independent of their surface composition (PArg

or HMWHA). The stability profiles of each LMWHA-containing formulation

and those of 0.65 charge-ratio containing HMWHA are not shown in the plot

because they either immediately aggregated after addition to the medium or

were larger than ~1000 nm. Differences in the stability of HMWHA- and

LMWHA-containing nanostructures may also be related to the specific

organization of HA and PArg on the nanoparticle surface. This would affect

the colloidal stability conferred by charge repulsion and steric hindrance. In

the case of the formulation of charge ratio 0.65 with HMWHA, its instability

could be attributed that this formulation shows the lowest magnitude in zeta

potential before the point of charge inversion, thus effectively perturbing the

stability of the systems mediated by charge repulsion. Importantly, in Figure

2 it is possible to appreciate that, from the beginning of the experiment (0 h),

all the formulations showed an increased size compared with the original one

evaluated in MilliQ water (Table 1). Considering that PBS presents a

significant quantity of salt, this can weaken and dissociate the anionic

interactions between the oppositely charged polymers leading to swelling of

the systems. Additionally, the salt ions could diffuse inside the nanosystems

and attracting, by osmotic forces, water inside the nanosystems also exerting

a possible swelling of the formulations.

Anexo 2

293

Figure 2: Stability profiles of HMWHA-PArg nanocarriers in phosphate-buffered saline pH 7.4 at 37°C; HA-PArg charge ratios: 0.325 (x), 0.975 (+), 1.3 ( ), 1.625 ( ) and 1.95 ( ) (mean ± S.D., n=3).

Finally, the stability of nanocarriers during long-term storage is an

important step for the adequate handling of the formulations. Figure 3 shows

the stability profiles of each formulation composed of HMWHA at 4 °C for 3

months. Every formulation was stable and showed no significant change in

particle size. Each system composed of LMWHA was also stable during

storage (data not shown).

Figure 3: Stability profiles of HMWHA-PArg nanocarriers in water during storage at 4°C; HA-PArg charge ratios: 0.325 (x), 0.65 ( ), 0.975 (+), 1.3 ( ), 1.625 ( ) and 1.95 ( ) (mean ± S.D., n=3).


294

As future work, we are planning to introduce macromolecular

hydrophilic drugs into the nanocarriers whose final targets could be solid

tumor cells (where CD-44 receptors are overexpressed), or mucosal surfaces

(that can avidly interact with both hyaluronic acid and polyarginine). More

concretely, oral peptide delivery, in which polyarginine has demonstrated to

be very promising, will be explored.

Conclusions

In conclusion, a nanoparticulate system composed of PArg and HA

was successfully prepared using an extremely mild process. Negatively and

positively charged nanoparticle formulations with surfaces composed

preferentially of HA or PArg, were obtained. Importantly, we demonstrated

that the molecular weight of HA is a crucial determinant of formulation

stability during mechanical isolation and in physiological conditions. This

knowledge is useful not only for systems comprising polyarginine and

hyaluronic acid but also for systems composed by other polymers. Further

studies testing the potential of these systems as mucoadhesive nanocarriers

for targeted drug delivery will be carried out, and the in vitro-in vivo

behavior of these systems will be also evaluated.

Acknowledgements

The authors acknowledge the Spanish Government for financial

support (Consolider-Ingenio CSD 2006-00012 and Xunta de Galicia PGIDIT

08CSA045209PR) and CONICYT for a scholarship to F.A. Oyarzun-

Ampuero.

Anexo 2

295

References

1. M. de la Fuente, N. Csaba, M. Garcia-Fuentes, M.J. Alonso,

Nanoparticles as protein and gene carriers to mucosal surfaces,

Nanomed. 3 (2008) 845-57.

2. V.M. Platt, F.C.Jr. Szoka, Anticancer therapeutics: targeting

macromolecules and nanocarriers to hyaluronan or CD44, a hyaluronan

receptor, Mol. Pharm. 5 (2008) 474-86.

3. F.A. Oyarzun-Ampuero, J. Brea, M.I. Loza, D. Torres, M.J. Alonso,


treatment of asthma, Int. J. Pharm. 381 (2009) 122-129.

4. Lundberg M.; Wikström S.; Johansson M. (2003). Cell Surface

Adherence and Endocytosis of Protein Transduction Domains. Mol.

Ther. 8 (2003) 143-150.

5. V.P. Torchilin, TAT peptide-mediated intracelular delivery of

pharmaceutical nanocarriers, Adv. Drug Deliv. Rev. 60 (2008) 548-58.

6. K. Lingnau, K. Riedl, A. von Gabain, IC31® and IC30, novel types of

vaccine adjuvant based on peptide delivery systems, Expert Rev.

Vaccines. 6 (2007) 741-746.

7. Z. Miklán, E. Orbán, G. Csík, G. Schlosser, A. Magyar, F. Hudecz,

New daunomycin-oligoarginine conjugates: synthesis, characterization,

and effect on human leukemia and human hepatoma cells, Biopolymers.

92 (2009) 489-501.

8. M. Miyamoto, H. Natsume, S. Iwata, K. Ohtake, M. Yamaguchi, D.

Kobayashi, K. Sugibayashi, M. Yamashina, Y. Morimoto, Improved

nasal absorption of drugs using poly-L-arginine: effects of concentration

and molecular weight of poly-L-arginine on the nasal absorption of

fluorescein isothiocyanate-dextran in rats, Eur. J. Pharm. Biopharm. 52

(2001) 21-30.


296

9. E.J. Kim, G. Shim, K. Kim, I.C. Kwon, Y.K. Oh, C.K. Shim,

Hyaluronic acid complexed to biodegradable poly-L-arginine for

targeted delivery of siRNA, J. Gene Med. 11 (2009) 791-803.

10. F.M. Goycoolea, G. Lollo, C. Remuñán-López, F. Quaglia, M.J. Alonso,

Chitosan-alginate blended nanoparticles as carriers for the transmucosal

delivery of macromolecules. Biomacromolecules. 10 (2009) 1736-1743.

Anexo 3

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Anexo 3

Patentes Solicitadas:

M.J. Alonso, D. Torres, G. Rivera-Rodríguez, F. Oyarzún-Ampuero, G.

Lollo, T. Gonzalo-Lázaro, M. García-Fuentes. Nanocápsulas con cubierta

polimérica. Número de solicitud P201130015 (Oficina Española de Patentes

y Marcas), fecha de recepción 10-01-2011.

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